Hugo and Russell's. Pharmaceutical. Microbiology. EDITED BY. Stephen P
Denyer. B Pharm PhD FRPharmS. Welsh School of Pharmacy. Cardiff University.
Hugo and Russell’s
Pharmaceutical Microbiology EDITED BY
Stephen P Denyer B Pharm PhD FRPharmS Welsh School of Pharmacy Cardiff University Cardiff
Norman A Hodges B Pharm PhD MRPharmS School of Pharmacy and Biomolecular Sciences Brighton University Lewes Road Brighton
Sean P Gorman BSc PhD MPS School of Pharmacy Queen’s University Belfast Medical Biology Centre University Road Belfast
SEVENTH EDITION
Blackwell Science
Hugo and Russell’s Pharmaceutical Microbiology
Hugo and Russell’s
Pharmaceutical Microbiology EDITED BY
Stephen P Denyer B Pharm PhD FRPharmS Welsh School of Pharmacy Cardiff University Cardiff
Norman A Hodges B Pharm PhD MRPharmS School of Pharmacy and Biomolecular Sciences Brighton University Lewes Road Brighton
Sean P Gorman BSc PhD MPS School of Pharmacy Queen’s University Belfast Medical Biology Centre University Road Belfast
SEVENTH EDITION
Blackwell Science
© 1977, 1980, 1983, 1987, 1992, 1998, 2004 by Blackwell Science Ltd a Blackwell Publishing company Blackwell Science, Inc., 350 Main Street, Malden, Massachusetts 02148-5020, USA Blackwell Publishing Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK Blackwell Science Asia Pty Ltd, 550 Swanston Street, Carlton, Victoria 3053, Australia The right of the Author to be identified as the Author of this Work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. First published 1977 Second edition 1980 Third edition 1983 Reprinted 1986 Fourth edition 1987 Reprinted 1989, 1991
Italian edition 1991 Fifth edition 1992 Reprinted 1993, 1994, 1995 Sixth edition 1998 Reprinted 1999, 2000, 2002, 2003 Seventh edition 2004
Library of Congress Cataloging-in-Publication Data Hugo and Russell’s pharmaceutical microbiology / edited by Stephen Denyer, Norman A. Hodges, Sean P. Gorman. — 7th ed. p. cm. Rev. ed. of: Pharmaceutical microbiology / edited by W.B. Hugo and A.D. Russell. Includes bibliographical references and index. ISBN 0-632-06467-6 1. Pharmaceutical microbiology. [DNLM: 1. Anti-Infective Agents. 2. Technology, Pharmaceutical. QV 250 H895 2004] I. Title: Pharmaceutical microbiology. II. Hugo, W. B. (William Barry) III. Denyer, S. P. IV. Hodges, Norman A.V,. Gorman, S. P. VI. Pharmaceutical microbiology. QR46.5.P48 2004 615¢.1¢01579 — dc22 2003024264 ISBN 0–632–06467–6 A catalogue record for this title is available from the British Library Set in Sabon 9.5/12 pt by SNP Best-set Typesetter Ltd., Hong Kong Printed and bound in the United Kingdom by Ashford Colour Press, Gosport Commissioning Editor: Maria Khan Managing Editor: Rupal Malde Production Editor: Fiona Pattison Production Controller: Kate Charman For further information on Blackwell Publishing, visit our website: http://www.blackwellpublishing.com
Contents
Contributors, vii Preface to Seventh Edition, ix Preface to First Edition, x Part 1: Biology of Microorganisms 1. Introduction to Pharmaceutical Microbiology, 3 Stephen Denyer, Norman Hodges and Sean Gorman 2. Fundamental Features of Microbiology, 9 Norman Hodges 3. Bacteria, 23 David Allison and Peter Gilbert 4. Fungi, 44 Kevin Kavanagh and Derek Sullivan 5. Viruses, 59 Jean-Yves Maillard and David Stickler 6. Protozoa, 82 Tim Paget 7. Principles of Microbial Pathogenicity and Epidemiology, 103 Peter Gilbert and David Allison Part 2: Antimicrobial Agents 8. Basic Aspects of the Structure and Functioning of the Immune System, 117 Mark Gumbleton and James Furr 9. Vaccination and Immunization, 138 Peter Gilbert and David Allison 10. Types of Antibiotics and Synthetic Antimicrobial Agents, 152 A Denver Russell
11. Laboratory Evaluation of Antimicrobial Agents, 187 JMB Smith 12. Mechanisms of Action of Antibiotics and Synthetic Anti-infective Agents, 202 Peter Lambert 13. Bacterial Resistance to Antibiotics, 220 Anthony Smith 14. Clinical Uses of Antimicrobial Drugs, 233 Roger Finch
Part 3: Microbiological Aspects of Pharmaceutical Processing 15. Ecology of Microorganisms as it Affects the Pharmaceutical Industry, 251 Elaine Underwood 16. Microbial Spoilage, Infection Risk and Contamination Control, 263 Rosamund Baird 17. Chemical Disinfectants, Antiseptics and Preservatives, 285 Sean Gorman and Eileen Scott 18. Non-Antibiotic Antibacterial Agents: Mode of Action and Resistance, 306 Stephen Denyer and A Denver Russell 19. Sterile Pharmaceutical Products, 323 James Ford 20. Sterilization Procedures and Sterility Assurance, 346 Stephen Denyer and Norman Hodges 21. Factory and Hospital Hygiene, 376 Robert Jones 22. Manufacture of Antibiotics, 387 Sally Varian
v
Contents
23. The Manufacture and Quality Control of Immunological Products, 398 Michael Corbel 24. Pharmaceutical Biotechnology, 416 Miguel Cámara
vi
25. Additional Applications of Microorganisms in the Pharmaceutical Sciences, 441 Denver Russell Index, 459
Contributors
Dr David Allison
Professor James Ford
Dr Robert Jones
School of Pharmacy and Pharmaceutical Sciences University of Manchester Oxford Road Manchester M13 9PL UK
School of Pharmacy and Chemistry Liverpool John Moores University Byrom Street Liverpool L3 3AF UK
School of Pharmacy and Biomedical Sciences University of Portsmouth St Michael’s Building White Swan Road Portsmouth PO1 2DT UK
Dr James Furr Dr Rosamund Baird Visiting Senior Lecturer School of Pharmacy and Pharmacology University of Bath Claverton Down Bath BA2 7AY UK
Dr Miguel Cámara Senior Lecturer in Molecular Microbiology Institute of Pharmaceutical Sciences School of Pharmaceutical Sciences University of Nottingham Nottingham NG7 2RD UK
Dr Michael Corbel National Institute for Biological Standards and Control Blanche Lane South Mimms Potters Bar Hertfordshire EN6 3QG UK
Professor Stephen Denyer Welsh School of Pharmacy Cardiff University Cardiff CF10 3XF UK
Professor Roger Finch Professor of Infectious Diseases Clinical Sciences Building University of Nottingham The City Hospital Nottingham NG5 1PB UK
Welsh School of Pharmacy Cardiff University King Edward VII Avenue Cardiff CF10 3XF Wales
Professor Peter Gilbert School of Pharmacy and Pharmaceutical Sciences University of Manchester Oxford Rd Manchester M13 9PL UK
Dr Kevin Kavanagh Head of Medical Mycology Unit Department of Biology National University of Ireland Maynooth Co. Kildare Ireland
Dr Peter Lambert Aston Pharmacy School Aston University Aston Triangle Birmingham B4 7ET UK
Professor Sean Gorman Professor of Pharmaceutical Microbiology School of Pharmacy The Queen’s University of Belfast Belfast BT9 7BL Northern Ireland
Dr Mark Gumbleton Welsh School of Pharmacy Cardiff University King Edward VII Avenue Cardiff CF10 3XF Wales
Dr Jean-Yves Maillard School of Pharmacy and Biomolecular Sciences University of Brighton Lewes Rd Brighton BN2 4GJ UK
Dr Tim Paget Department of Biological Sciences University of Hull Hull HU6 7RX UK
Dr Norman Hodges Principal Lecturer in Pharmaceutical Microbiology School of Pharmacy and Biomolecular Sciences University of Brighton Lewes Road Brighton BN2 4GJ UK
Professor A Denver Russell Welsh School of Pharmacy Cardiff University King Edward VII Avenue Cardiff CF10 3XF Wales
Dr Eileen Scott School of Pharmacy The Queen’s University of Belfast Belfast BT9 7BL Northern Ireland
vii
Contributors
Dr Anthony Smith
Dr David Stickler
Dr Elaine Underwood
Department of Pharmacy and Pharmacology University of Bath (5 West — 2.18) Claverton Down Bath BA2 7AY UK
School of Biosciences Cardiff University Main Building Museum Avenue PO Box 915 Cardiff CF10 3TL Wales
SMA Nutrition Huntercomb Lane South Taplow Maidenhead Berks SL6 0PH UK
Professor JMB (Sandy) Smith Head of Department of Microbiology Otago School of Medical Sciences University of Otago Dunedin New Zealand
viii
Dr Sally Varian Dr Derek Sullivan Microbiology Research Unit School of Dental Science Trinity College Dublin 2 Ireland
Consultant Ulverston Cumbria LA12 8PT UK
Preface to the Seventh edition
We were much honoured to be recommended by Professor A.D. Russell to act as editors for the 7th edition of Pharmaceutical Microbiology. All three of us have used this textbook in its various editions throughout our careers as teachers and researchers, and we recognize the important role it fulfils. As might be anticipated when a new editorial team is in place, a substantial number of changes have been made. Well over half the chapters have new authors or co-authors. We also use Chapter 1 to give a rationale for the scope and content of the book, emphasizing the interrelated character of the discipline of pharmaceutical microbiology. In addition, by combining and reorganizing chapters, by introducing new material and through a revised page format we have tried to provide readers with a distinctive 7th edition.
We must thank our contributors for their willing collaboration in this enterprise, especially Professor Russell for his continuing contributions, and our publishers for their support and expertise. Finally, this addition is a tribute to the farsightedness of A.D. Russell and W.B. Hugo who took up the challenge in 1977 to produce a popular and concise read for pharmacy students required to study pharmaceutical microbiology. We are delighted that this current edition recognizes these origins by continuing the association with Hugo and Russell in its revised title. S.P. Denyer S.P. Gorman N.A. Hodges
ix
Preface to the First Edition
When we were first approached by the publishers to write a textbook on pharmaceutical microbiology to appear in the spring of 1977, it was felt that such a task could not be accomplished satisfactorily in the time available. However, by a process of combined editorship and by invitation to experts to contribute to the various chapters this task has been accomplished thanks to the cooperation of our collaborators. Pharmaceutical microbiology may be defined as that part of microbiology which has a special bearing on pharmacy in all its aspects. This will range from the manufacture and quality control of pharmaceutical products to an understanding of the mode of action of antibiotics. The full extent of microbiology on the pharmaceutical area may be judged from the chapter contents. As this book is aimed at undergraduate pharmacy students (as well as microbiologists entering the pharmaceutical industry) we were under constraint to limit the length of the book to retain it in a defined price range. The result is to be found in
x
the following pages. The editors must bear responsibility for any omissions, a point which has most concerned us. Length and depth of treatment were determined by the dictate of our publishers. It is hoped that the book will provide a concise reading for pharmacy students (who, at the moment, lack a textbook in this subject) and help to highlight those parts of a general microbiological training which impinge on the pharmaceutical industry. In conclusion, the editors thank most sincerely the contributors to this book, both for complying with our strictures as to the length of their contribution and for providing their material on time, and our publishers for their friendly courtesy and efficiency during the production of this book. We also wish to thank Dr H.J. Smith for his advice on various chemical aspects, Dr M.I. Barnett for useful comments on reverse osmosis, and Mr A. Keall who helped with the table on sterilization methods. W.B. Hugo A.D. Russell
Part 1
Biology of Microorganisms
Chapter 1
Introduction to pharmaceutical microbiology Stephen Denyer, Norman Hodges and Sean Gorman 1 Microorganisms and medicines
2 The scope and content of the book
1 Microorganisms and medicines
the most consistently successful and important industries in many countries, not only in the traditional strongholds of North America, Western Europe and Japan but, increasingly, in Eastern Europe, the Indian subcontinent and the Far East. Worldwide sales of medicines and medical devices are estimated to have exceeded $US 401 billion (approximately £250 billion) in 2002, and this figure is rising by 8% per annum. In the UK alone, the value of pharmaceutical exports is currently £10.03 billion each year, a figure that translates to more than £150 000 for each employee in the industry. The growth of the pharmaceutical industry in recent decades has been paralleled by rising standards for product quality and more rigorous regulation of manufacturing procedures. In order to receive a manufacturing licence, a modern medicine must be shown to be effective, safe and of good quality. Most medicines consist of an active ingredient that is formulated with a variety of other materials (excipients) that are necessary to ensure that the medicine is effective, and remains stable, palatable and safe during storage and use. While the efficacy and safety aspects of the active ingredient are within the domain of the pharmacologist and toxicologist, respectively, many other disciplines contribute to the efficacy, safety and quality of the manufactured product as a whole. Analytical chemists and pharmacists take lead responsibility for ensuring that the components of the medicine are present in the correct physical form and concentration, but quality is not judged solely on the physicochemical properties of the product: microorganisms also have the potential to influence efficacy and safety. It is obvious that medicines contaminated with potentially pathogenic (disease-causing) micro-
Despite continuing poverty in many parts of the world and the devastating effects of HIV and AIDS infection on the African continent and elsewhere, the health of the world’s population is progressively improving. This is reflected in the increase in life expectancy that has been recorded for the great majority of the countries reporting statistics to the World Health Organization over the last 40 years. In Central America, for example, the life expectancy has increased from 55 years in 1960 to 71 years in 2000, and the increase in North (but not subSaharan) Africa is even greater, from 47 to 68 years. Much of this improvement is due to better nutrition and sanitation, but improved health care and the greater availability of effective medicines with which to treat common diseases are also major contributing factors. Substantial inroads have been made in the prevention and treatment of cancer, cardiovascular disease and other major causes of death in Western society, and of infections and diarrhoeal disease that remain the big killers in developing countries. Several infectious diseases have been eradicated completely, and others from substantial parts of the world. The global eradication of smallpox in 1977 is well documented, but 2002 saw three of the world’s continents declared free of polio, and the prospects are good for the total elimination of polio, measles and Chagas disease. The development of the many vaccines and other medicines that have been so crucial to the improvement in world heath has been the result of the large investment in research by the major international pharmaceutical companies. This has led to the manufacture of pharmaceuticals becoming one of
3
Chapter 1
organisms are a safety hazard, so medicines administered by vulnerable routes (e.g. injections) or to vulnerable areas of the body (e.g. the eyes) are manufactured as sterile products. What is less predictable is that microorganisms can, in addition to initiating infections, cause product spoilage by chemically decomposing the active ingredient or the excipients. This may lead to the product being under-strength, physically or chemically unstable or possibly contaminated with toxic materials. Thus, it is clear that pharmaceutical microbiology must encompass the subjects of sterilization and preservation against microbial spoilage, and a pharmacist with responsibility for the safe, hygienic manufacture and use of medicines must know where microorganisms arise in the environment, i.e. the sources of microbial contamination, and the factors that predispose to, or prevent, product spoilage. In these respects, the pharmaceutical microbiologist has a lot in common with food and cosmetics microbiologists, and there is substantial scope for transfer of knowledge between these disciplines. Disinfection and the properties of chemicals (biocides) used as antiseptics, disinfectants and preservatives are subjects of which pharmacists and other persons responsible for the manufacture of medicines should have a knowledge, both from the perspective of biocide use in product formulation and manufacture, and because antiseptics and disinfectants are pharmaceutical products in their own right. However, they are not the only antimicrobial substances that are relevant to medicine; antibiotics are of major importance and represent a product category that regularly features among the top five most frequently prescribed. The term ‘antibiotic’ is used in several different ways: originally an antibiotic was defined as a naturally occurring substance that was produced by one microorganism that inhibited the growth of, or killed, other microorganisms, i.e. an antibiotic was a natural product, a microbial metabolite. More recently the term has come to encompass certain synthetic agents that are usually used systemically (throughout the body) to treat infection. A knowledge of the manufacture, quality control and, in the light of current concerns about resistance of microorganisms, the use of antibiotics, are other areas of knowledge that 4
contribute to the discipline of pharmaceutical microbiology. Commercial antibiotic production began with the manufacture of penicillin in the 1940s, and for many years antibiotics were the only significant example of a medicinal product that was made using microorganisms. Following the adoption in the 1950s of microorganisms to facilitate the manufacture of steroids and the development of recombinant DNA technology in the last three decades of the 20th century, the use of microorganisms in the manufacture of medicines has gathered great momentum. It led to more than 100 biotechnologyderived products on the market by the new millennium and another 300 or more in clinical trials. While it is true to say that traditionally the principal pharmaceutical interest in microorganisms is that of controlling them, exploiting microbial metabolism in the manufacture of medicines is a burgeoning area of knowledge that will become increasingly important, not only in the pharmacy curriculum but also in those of other disciplines employed in the pharmaceutical industry. Table 1.1 summarizes these benefits and uses of microorganisms in pharmaceutical manufacturing, together with the more widely recognized hazards and problems that they present. Looking ahead to the early decades of the 21st century, it is clear that an understanding of the physiology and genetics of microorganisms will also become more important, not just in the production of new therapeutic agents but in the understanding of infections and other diseases. Several of the traditional diseases that were major causes of death before the antibiotic era, e.g. tuberculosis and diphtheria, are now re-emerging in resistant form — even in developed countries — adding to the problems posed by infections in which antibiotic resistance has long been a problem, and those like Creutzfeldt–Jakob disease, West Nile virus and severe acute respiratory syndrome (SARS) that have only been recognized in recent years. Not only has the development of resistance to established antibiotics become a challenge, so too has the ability of microorganisms to take advantage of changing practices and procedures in medicine and surgery. Microorganisms are found almost everywhere in our surroundings and they possess
Related study topics Good manufacturing practice Industrial ‘fermentation’ technology Microbial genetics
Quality control of immunological products Assay methods
Ames mutagenicity test
Benefits or uses
The manufacture of: antibiotics steroids therapeutic enzymes polysaccharides products of recombinant DNA technology
Use in the production of vaccines
As assay organisms to determine antibiotic, vitamin and amino acid concentrations
To detect mutagenic or carcinogenic activity
Table 1.1 Microorganisms in pharmacy: benefits and problems
Enumeration, identification and detection as above, plus Characteristics, selection and testing of antimicrobial preservatives Immunology and infectious diseases Characteristics, selection and use of vaccines and antibiotics Use of biocides in infection and contamination control Control of antibiotic resistance Bacterial structure Pyrogen and endotoxin testing Microbial genetics
Cause infectious and other diseases
Cause pyrogenic reactions (fever) when introduced into the body even in the absence of infection Provide a reservoir of antibiotic resistance genes
Non- sterile medicines: Enumeration of microorganisms in the manufacturing environment (environmental monitoring) and in raw materials and manufactured products Identification and detection of specific organisms Sterile medicines: Sterilization methods Sterilization monitoring and validation procedures Sterility testing Assessment and calculation of sterility assurance Aseptic manufacture
Study topic
May contaminate nonsterile and sterile medicines with a risk of product deterioration
May contaminate nonsterile and sterile medicines with a risk of infection
Harmful effects
Introduction to pharmaceutical microbiology
5
Chapter 1
the potential to reproduce extremely rapidly; it is quite possible for cell division to occur every 20 minutes under favourable conditions. These characteristics mean that they can adapt readily to a changing environment and colonize new niches. One feature of modern surgery is the everincreasing use of plastic, ceramic and metal devices that are introduced into the body for a wide variety of purposes, including the commonly encountered urinary or venous catheters and the less common intra-ocular lenses, heart valves, pacemakers and hip prostheses. Many bacteria have the potential to produce substances or structures that help them to attach to these devices, even while combating the immune system of the body. Thus, colonization often necessitates removal and replacement of the device in question — often leading to great discomfort for the patient and substantial monetary cost to the health-care service. It has recently been estimated that, on average, a hospital-acquired infection results in an extra 14 days in hospital, a 10% increase in the chance of dying and more than £3000 additional expenditure on health care. The development of strategies for eliminating, or at least restricting, the severity or consequences of these device-related infections is a challenge for pharmacists and microbiologists within the industry, and for many other health-care professionals. In addition to an improved understanding of the mechanisms of antibiotic resistance, of the links between antibiotic resistance and misuse, and of the factors influencing the initiation of infections in the body, our insights into the role of microorganisms in other disease states have broadened significantly in recent years. Until about 1980 it was probably true to say that there was little or no recognition of the possibility that microorganisms might have a role to play in human diseases other than clear-cut infections. In recent years, however, our perception of the scope of microorganisms as agents of disease has been changed by the discovery that Helicobacter pylori is intimately involved in the development of gastric or duodenal ulcers and stomach cancer; by the findings that viruses can cause cancers of the liver, blood and cervix; and by the suspected involvement of microorganisms in diverse conditions like parkinsonism and Alzheimer’s disease. 6
Clearly, a knowledge of the mechanisms whereby microorganisms are able to resist antibiotics, colonize medical devices and cause or predispose humans to other disease states is essential in the development not only of new antibiotics, but of other medicines and health-care practices that miminize the risks of these adverse situations developing.
2 The scope and content of the book Criteria and standards for the microbiological quality of medicines depend upon the route of administration of the medicine in question. The vast majority of medicines that are given by mouth or placed on the skin are non-sterile, i.e. they may contain some microorganisms (within limits on type and concentration), whereas all injections and ophthalmic products must be sterile, i.e. they contain no living organisms. Products for other anatomical sites (e.g. nose, ear, vagina and bladder) are often sterile but not invariably so (Chapter 19). The microbiological quality of non-sterile medicines is controlled by specifications defining the concentration of organisms that may be present and requiring the absence of specific, potentially hazardous organisms. Thus the ability to identify the organisms present, to detect those that are prohibited from particular product categories and to enumerate microbial contaminants in the manufacturing environment, raw materials and finished product are clearly skills that a pharmaceutical microbiologist should possess (Chapters 2–6). So, too, is a familiarity with the characteristics of antimicrobial preservatives that may be a component of the medicine required to minimize the risk of microbial growth and spoilage during storage and use by the patient (Chapters 16 and 17). For a sterile product the criterion of quality is simple; there should be no detectable microorganisms whatsoever. The product should, therefore, be able to pass a test for sterility, and a knowledge of the procedures and interpretation of results of such tests is an important aspect of pharmaceutical microbiology (Chapter 20). Injections are also subject to a test for pyrogens; these are substances that cause a rise in body temperature when introduced
Introduction to pharmaceutical microbiology
into the body. Strictly speaking, any substance which causes fever following injection is a pyrogen, but in reality the vast majority are of bacterial origin, and it is for this reason that the detection, assay and removal of bacterial pyrogens (endotoxins) are considered within the realm of microbiology (Chapter 19). Sterile medicines may be manufactured by two different strategies. The most straightforward and preferred option is to make the product, pack it in its final container and sterilize it by heat, radiation or other means (terminal sterilization, Chapter 20). The alternative is to manufacture the product from sterile ingredients under conditions that do not permit the entry of contaminating organisms (aseptic manufacture, Chapters 15 and 21); this latter option is usually selected when the ingredients or physical form of the product render it heat- or radiation-sensitive. Those responsible for the manufacture of sterile products must be familiar with the sterilization or aseptic manufacturing procedures available for different product types, and those who have cause to open, use or dispense sterile products (in a hospital pharmacy, for example) should be aware of the aseptic handling procedures to be adopted in order to minimize the risk of product contamination. The spoilage of medicines as a result of microbial contamination, although obviously undesirable, has as its main consequence financial loss rather than ill health on the part of the patient. The other major problem posed by microbial contamination of medicines, that of the risk of initiating infection, although uncommon, is far more important in terms of risk to the patient and possible loss of life (Chapters 7 and 16). Infections arising by this means also have financial implications, of course, not only in additional treatment costs but in terms of product recalls, possible litigation and damage to the reputation of the manufacturer. The range of antimicrobial drugs used to prevent and treat microbial infections is large; for example, a contemporary textbook of antimicrobial chemotherapy lists no fewer than 43 different cephalosporin antibiotics that were already on the market or the subject of clinical trials at the time of publication. Not only are there many antibiotic products, but increasingly, these products really
have properties that make them unique. It is far more difficult now than it was, say, 20 years ago, for a manufacturer to obtain a licence for a ‘copycat’ product, as licensing authorities now emphasize the need to demonstrate that a new antibiotic (or any new medicine) affords a real advantage over established drugs. Because of this range and diversity of products, pharmacists are now far more commonly called upon to advise on the relative merits of the antibiotics available to treat particular categories of infection than was the case hitherto (Chapters 10, 12 and 14). A prerequisite to provide this information is a knowledge not only of the drug in question, but the infectious disease it is being used to treat and the factors that might influence the success of antibiotic therapy in that situation (Chapter 7). While there was a belief among some commentators a generation ago that infectious disease was a problem that was well on the way to permanent resolution owing to the development of effective vaccines and antibiotics, such complacency has now completely disappeared. Although cardiovascular and malignant diseases are more frequent causes of death in many developed countries, infectious diseases remain of paramount importance in many others, so much so that the five leading infections — respiratory, HIV/AIDS, diarrhoeal disease, tuberculosis and malaria, accounted for 11.5 million deaths in 1999. The confidence that antibiotics would be produced to deal with the vast majority of infections has been replaced by a recognition that the development of resistance to them is likely to substantially restrict their value in the control of certain infections (Chapter 13). Resistance to antibiotics has increased in virtually all categories of pathogenic microorganisms and is now so prevalent that there are some infections and some organisms for which, it is feared, there will soon be no effective antibiotics. It has been estimated that the annual cost of treating hospital-acquired infections may be as high as $4 billion in the USA alone. The scale and costs of the problem are such that increasing attention is being paid to infection control procedures that are designed to minimize the risk of infection being transmitted from one patient to another within a hospital. The properties of disinfectants and antiseptics, the measurement of their antimicrobial activity and the factors influenc7
Chapter 1
ing their selection for use in hospital infection control strategies or contamination control in the manufacturing setting are topics with which both pharmacists and industrial microbiologists should be familiar (Chapters 11 and 18). It has long been recognized that microorganisms are valuable, if not essential, in the maintenance of our ecosystems. Their role and benefits in the carbon and nitrogen cycles in terms of recycling dead plant and animal material and in the fixation of atmospheric nitrogen are well understood. The uses of microorganisms in the food, dairy and brewing industries are also well established, but until the late 20th century advances in genetics, immunology and biotechnology, their benefits and uses in the pharmaceutical industry were far more modest. For many years the production of antibiotics (Chapter 22) and microbial enzyme-mediated production of steroids were the only significant pharmaceutical examples of the exploitation of metabolism of microorganisms. The value of these applications, both in monetary and health-care terms has been immense. Antibiotics currently have an estimated world market value of $25 billion and by this criterion they are surpassed as products of biotechnology only by cheese and alcoholic beverages, but the benefits they afford in terms of improved health and life expectancy are incalculable. The discovery of the anti-inflammatory effects of corticosteroids had a profound impact on the treatment of rheumatoid arthritis in the 1950s, but it was the use of enzymes possessed by common fungi that made cortisone widely available to rheumatism sufferers. The synthesis of cortisone by traditional chemical methods involved 31 steps, gave a yield of less than 0.2% of the starting material and resulted in a product costing, even in 1950s terms, $200 per gram. Exploiting
8
microbial enzymes reduced the synthesis to 11 steps and the cost rapidly fell to $6 per gram. Apart from these major applications, however, the uses of microorganisms in the manufacture of medicines prior to 1980 were very limited. Enzymes were developed for use in cancer chemotherapy (asparaginase) and to digest blood clots (streptokinase), and polysaccharides also found therapeutical applications (e.g. dextran — used as a plasma expander). These were of relatively minor importance, however, compared with the products that followed the advances in recombinant DNA technology in the 1970s. This technology permitted human genes to be inserted into microorganisms, which were thus able to manufacture the gene products far more efficiently than traditional methods of extraction from animal or human tissues. Insulin, in 1982, was the first therapeutic product of DNA technology to be licensed for human use, and it has been followed by human growth hormone, interferon, blood clotting factors and many other products. DNA technology has also permitted the development of vaccines which, like that for the prevention of hepatitis B, use genetically engineered surface antigens rather than whole natural virus particles, so these vaccines are more effective and safer than those produced by traditional means (Chapters 9 and 23). All these developments, together with miscellaneous applications in the detection of mutagenic and carcinogenic activity in drugs and chemicals and in the assay of antibiotics, vitamins and amino acids (Chapter 25), have ensured that the role of microorganisms in the manufacture of medicines is now well recognized, and that a basic knowledge of immunology (Chapter 8), gene cloning and other biotechnology disciplines (Chapter 24) is an integral part of pharmaceutical microbiology.
Chapter 2
Fundamental features of microbiology Norman Hodges 1 Introduction 1.1 Viruses, viroids and prions 1.2 Prokaryotes and eukaryotes 1.2.1 Bacteria and archaea 1.2.2 Fungi 1.2.3 Protozoa 2 Naming of microorganisms 3 Microbial metabolism 4 Microbial cultivation 4.1 Culture media
4.2 Cultivation methods 4.3 Planktonic and sessile growth 5 Enumeration of microorganisms 6 Microbial genetics 6.1 Bacteria 6.2 Eukaryotes 6.3 Genetic variation and gene expression 7 Pharmaceutical importance of the major categories of microorganisms
1 Introduction
absent. Viruses are incapable of independent replication as they do not contain the enzymes necessary to copy their own nucleic acids; as a consequence, all viruses are intracellular parasites and are reproduced using the metabolic capabilities of the host cell. A great deal of variation is observed in shape (helical, linear or spherical), size (20–400 nm) and nucleic acid composition (single- or doublestranded, linear or circular RNA or DNA), but almost all viruses are smaller than bacteria and they cannot be seen with a normal light microscope; instead they may be viewed using an electron microscope which affords much greater magnification. Viroids (virusoids) are even simpler than viruses, being infectious particles comprising single-stranded RNA without any associated protein. Those that have been described are plant pathogens, and, so far, there are no known human pathogens in this category. Prions are unique as infectious agents in that they contain no nucleic acid. A prion is an atypical form of a mammalian protein that can interact with a normal protein molecule and cause it to undergo a conformational change so that it, in turn, becomes a prion and ceases its normal function. Prions are the agents responsible for transmissible spongiform encephalopathies, e.g. Creutzfeldt– Jakob disease (CJD) and bovine spongiform encephalopathy (BSE). They are the simplest and most
Microorganisms differ enormously in terms of their shape, size and appearance and in their genetic and metabolic characteristics. All these properties are used in classifying microorganisms into the major groups with which many people are familiar, e.g. bacteria, fungi, protozoa and viruses, and into the less well known categories like chlamydia, rickettsia and mycoplasmas. The major groups are the subject of individual chapters immediately following this, so the purpose here is not to describe any of them in great detail but to summarize their features so that the reader may better understand the distinctions between them. A further aim of this chapter is to avoid undue repetition of information in the early part of the book by considering such aspects of microbiology as cultivation, enumeration and genetics that are common to some, or all, of the various types of microorganism. 1.1 Viruses, viroids and prions Viruses do not have a cellular structure. They are particles composed of nucleic acid surrounded by protein; some possess a lipid envelope and associated glycoproteins, but recognizable chromosomes, cytoplasm and cell membranes are invariably
9
Chapter 2
recently recognized agents of infectious disease, and are important in a pharmaceutical context owing to their extreme resistance to conventional sterilizing agents like steam, gamma radiation and disinfectants (Chapter 18). 1.2 Prokaryotes and eukaryotes The most fundamental distinction between the various microorganisms having a cellular structure (i.e. all except those described in section 1.1 above) is their classification into two groups — the prokaryotes and eukaryotes — based primarily on their cellular structure and mode of reproduction. Expressed in the simplest possible terms, prokaryotes are the bacteria and archaea (see section 1.2.1), and eukaryotes are all other cellular microorganisms, e.g. fungi, protozoa and algae. The crucial difference between these two types of cell is the possession by the eukaryotes of a true cell nucleus in which the chromosomes are separated from the cytoplasm by a nuclear membrane. The prokaryotes have no true nucleus; they normally possess just a single chromosome that is not separated from the other cell contents by a membrane. Other major distinguishing features of the two groups are that prokaryotes are normally haploid (possess only one
copy of the set of genes in the cell) and reproduce asexually; eukaroyotes, by contrast, are usually diploid (possess two copies of their genes) and normally have the potential to reproduce sexually. The capacity for sexual reproduction confers the major advantage of creating new combinations of genes, which increases the scope for selection and evolutionary development. The restriction to an asexual mode of reproduction means that the organism in question is heavily reliant on mutation as a means of creating genetic variety and new strains with advantageous characteristics, although many bacteria are able to receive new genes from other strains or species (see section 6.1 and Chapter 3). Table 2.1 lists some distinguishing features of the prokaryotes and eukaryotes. 1.2.1 Bacteria and archaea Bacteria are essentially unicellular, although some species arise as sheathed chains of cells. They possess the properties listed under prokaryotes in Table 2.1, but, like viruses and other categories of microorganisms, exhibit great diversity of form, habitat, metabolism, pathogenicity and other characteristics. The bacteria of interest in pharmacy and medicine belong to the group known as the
Table 2.1 Distinguishing features of prokaryotes and eukaryotes Characteristic
Eukaryotes
Prokaryotes
Size Location of chromosomes
Normally > 10 µm Within a true nucleus separated from the cytoplasm by a nuclear membrane Exhibit mitosis and meiosis Present Asexual or sexual reproduction >1 May be present Sterols present Cell walls (when present) usually contain cellulose or chitin but not peptidoglycan Cytoplasmic ribosomes are 80S Structurally complex Absent Cilia Poly-b-hydroxybutyrate absent
Typically 1–5 µm In the cytoplasm, usually attached to the cell membrane Mitosis and meiosis are absent Absent Normally asexual reproduction 1 Absent Sterols absent Walls usually contain peptidoglycan
Nuclear division Nucleolus Reproduction Chromosome number Mitochondria and chloroplasts Cell membrane composition Cell wall composition Ribosomes Flagella Pili Fimbriae Storage compounds
10
Ribosomes are smaller, usually 70S Structurally simple Present Present Poly-b-hydroxybutyrate often present
Fundamental features of microbiology
eubacteria. The other subdivision of prokaryotes, the archaea, have little or no pharmaceutical importance and largely comprise organisms capable of living in extreme environments (e.g. high temperatures, extreme salinity or pH) or organisms exhibiting specialized modes of metabolism (e.g. by deriving energy from sulphur or iron oxidation or the production of methane). The eubacteria are typically rod-shaped (bacillus), spherical (cocci), curved or spiral cells of approximately 0.5–5.0 mm (longest dimension) and are divided into two groups designated Gram-positive and Gram-negative according to their reaction to a staining procedure developed in 1884 by Christian Gram (see Chapter 3). Although all the pathogenic species are included within this category there are very many other eubacteria that are harmless or positively beneficial. Some of the bacteria that contaminate or cause spoilage of pharmaceutical materials are saprophytes, i.e. they obtain their energy by decomposition of animal and vegetable material, while many could also be described as parasites (benefiting from growth on or in other living organisms without causing detrimental effects) or pathogens (parasites damaging the host). Rickettsia and chlamydia are types of bacteria that are obligate intracellular parasites, i.e. they are incapable of growing outside a host cell and so cannot easily be cultivated in the laboratory. Most bacteria of pharmaceutical and medical importance possess cell walls (and are therefore relatively resistant to osmotic stress), grow well at temperatures between ambient and human body temperature, and exhibit wide variations in their requirement for, or tolerance of, oxygen. Strict aerobes require atmospheric oxygen, but for strict anaerobes oxygen is toxic. Many other bacteria would be described as facultative anaerobes (normally growing best in air but can grow without it) or micro-aerophils (preferring oxygen concentrations lower than those in normal air). 1.2.2 Fungi Fungi are eukaryotes and therefore differ from bacteria in the ways described in Table 2.1 and are structurally more complex and varied in appearance. Fungi are considered to be non-photosynthe-
sizing plants, and the term fungus covers both yeasts and moulds, although the distinction between these two groups is not always clear. Yeasts are normally unicellular organisms that are larger than bacteria (typically 5–10 mm) and divide either by a process of binary fission (see section 4.2 and Fig. 2.1a) or budding (whereby a daughter cell arises as a swelling or protrusion from the parent that eventually separates to lead an independent existence, Fig. 2.1b). Mould is an imprecise term used to describe fungi that do not form fruiting bodies visible to the naked eye, thus excluding toadstools and mushrooms. Most moulds consist of a tangled mass (mycelium) of filaments or threads (hyphae) which vary between 1 and > 50 mm wide (Fig. 2.1c); they may be differentiated for specialized functions, e.g. absorption of nutrients or reproduction. Some fungi may exhibit a unicellular (yeast-like) or mycelial (mould-like) appearance depending upon cultivation conditions. Although fungi are eukaryotes that should, in theory, be capable of sexual reproduction, there are some species in which this has never been observed. Most fungi are saprophytes with relatively few having pathogenic potential, but their ability to form spores that are resistant to drying makes them important as contaminants of pharmaceutical raw materials, particularly materials of vegetable origin. 1.2.3 Protozoa Protozoa are eukaryotic, predominantly unicellular microorganisms that are regarded as animals rather than plants, although the distinction between protozoa and fungi is not always clear and there are some organisms whose taxonomic status is uncertain. Many protozoa are free-living motile organisms that occur in water and soil, although some are parasites of plants and animals, including humans, e.g. the organisms responsible for malaria and amoebic dysentery. Protozoa are not normally found as contaminants of raw materials or manufactured medicines and the relatively few that are of pharmaceutical interest owe that status primarily to their potential to cause disease.
11
Chapter 2
Fig. 2.1 (a) A growing culture of Bacillus megaterium in which cells about to divide by binary fission display constrictions (arrowed) prior to separation. (b) A growing culture of the yeast Saccharomyces cerevisiae displaying budding (arrowed). (c) The mould Mucor plumbeus exhibiting the typical appearance of a mycelium in which masses of asexual zygospores (arrowed) are formed on specialized hyphae. (d) The bacterium Streptomyces rimosus displaying the branched network of filaments that superficially resembles a mould mycelium. (e) The typical appearance of an overnight agar culture of Micrococcus luteus inoculated to produce isolated colonies (arrowed). (f) A single colony of the mould Aspergillus niger in which the actively growing periphery of the colony (arrowed) contrasts with the mature central region where pigmented asexual spores have developed.
2 Naming of microorganisms Microorganisms, just like other organisms, are normally known by two names: that of the genus (plural = genera) and that of the species. The former 12
is normally written with an upper case initial letter and the latter with a lower case initial letter, e.g. Staphylococcus aureus or Escherichia coli. These may be abbreviated by shortening the name of the genus provided that the shortened form is
Fundamental features of microbiology
unambiguous, e.g. Staph. aureus, E. coli. Both the full and the shortened names are printed in italics to designate their status as proper names (in old books, theses or manuscripts they might be in roman type but underlined). The species within a genus are sometimes referred to by a collective name, e.g. staphylococci or pseudomonads, and neither these names, nor names describing groups of organisms from different genera, e.g. coliforms, are italicized or spelt with an upper case initial letter.
3 Microbial metabolism As in most other aspects of their physiology, microorganisms exhibit marked differences in their metabolism. While some species can obtain carbon from carbon dioxide and energy from sunlight or the oxidation of inorganic materials like sulphides, the vast majority of organisms of interest in pharmacy and medicine are described as chemoheterotrophs — they obtain carbon, nitrogen and energy by breaking down organic compounds. The chemical reactions by which energy is liberated by digestion of food materials are termed catabolic reactions, while those that use the liberated energy to make complex cellular polymers, proteins, carbohydrates and nucleic acids, are called anabolic reactions. Food materials are oxidized in order to break them down and release energy from them. The term oxidation is defined as the removal or loss of electrons, but oxidation does not invariably involve oxygen, as a wide variety of other molecules can accept electrons and thus act as oxidizing agents. As the oxidizing molecule accepts the electrons, the other molecule in the reaction that provides them is simultaneously reduced. Consequently, oxidation and reduction are invariably linked and such reactions are often termed redox reactions. The term redox potential is also used, and this indicates whether oxidizing or reducing conditions prevail in a particular situation, e.g. in a body fluid or a culture medium. Anaerobic organisms prefer low redox potentials (typically zero to -200 mV or less) while aerobes thrive in high redox potential environments (e.g. zero to +200 mV or more).
There are marked similarities in the metabolic pathways used by pathogenic bacteria and by mammals. Many bacteria use the same process of glycolysis that is used by humans to begin the breakdown of glucose and the release of energy from it. Glycolysis describes the conversion of glucose, through a series of reactions, to pyruvic acid, and it is a process for which oxygen is not required, although glycolysis is undertaken by both aerobic and anaerobic organisms. The process releases only a relatively small amount of the energy stored in a sugar molecule, and aerobic microorganisms, in common with mammals, release much more of the energy by aerobic respiration. Oxygen is the molecule at the end of the sequence of respiratory reactions that finally accepts the electrons and allows the whole process to proceed, but it is worth noting that many organisms can also undertake anaerobic respiration, which uses other final electron acceptors, e.g. nitrate or fumarate. As an alternative to respiration many microorganisms use fermentation as a means of releasing more energy from sugar; fermentation is, by definition, a process in which the final electron acceptor is an organic molecule. The term is widely understood to mean the production by yeast of ethanol and carbon dioxide from sugar, but in fact many organisms apart from yeasts can undertake fermentation and the process is not restricted to common sugar (sucrose) as a starting material or to ethanol and carbon dioxide as metabolic products. Many pathogenic bacteria are capable of fermenting several different sugars and other organic materials to give a range of metabolic products that includes acids (e.g. lactic, acetic and propionic), alcohols (e.g. ethanol, propanol, butanediol) and other commercially important materials like the solvents acetone and butanol. Fermentation is, like glycolysis, an anaerobic process, although the term is commonly used in the pharmaceutical and biotechnology industries to describe the manufacture of a wide range of substances by microorganisms where the biochemical process is neither fermentative nor even anaerobic, e.g. many textbooks refer to antibiotic fermentation, but the production vessels are usually vigorously aerated and far from anaerobic. Microorganisms are far more versatile than mammals with respect to the materials that they can use 13
Chapter 2
as foods and the means by which those foods are broken down. Some pathogenic organisms can grow on dilute solutions of mineral salts and sugar (or other simple molecules like glycerol, lactic or pyruvic acids), while others can obtain energy from rarely encountered carbohydrates or by the digestion of proteins or other non-carbohydrate foods. In addition to accepting a wide variety of food materials, many microorganisms can use alternative metabolic pathways to break the food down depending on the environmental conditions, e.g. facultative anaerobes can switch from respiration to fermentation if oxygen supplies are depleted. It is partly this ability to switch to different metabolic pathways that explains why none of the major antibiotics work by interfering with the chemical reactions microorganisms use to metabolize their food. It is a fundamental principle of antibiotic action that the drug must exploit a difference in metabolism between the organism to be killed and the human host; without such a difference the antibiotic would be very toxic to the patient too. However, not only do bacteria use metabolic pathways for food digestion that are similar to our own, many of them would have the ability to switch to an alternative energy-producing pathway if an antibiotic was developed that interfered with a reaction that is unique to bacteria. The metabolic products that arise during the period when a microbial culture is actually growing are termed primary metabolites, while those that are produced after cell multiplication has slowed or stopped, i.e. in the ‘stationary phase’ (see Chapter 3), are termed secondary metabolites. Ethanol is a primary metabolite of major commercial importance although it is only produced in large quantities by some species of yeast. More common than ethanol as primary metabolites are organic acids, so it is a common observation that the pH of a culture progressively falls during growth, and many organisms further metabolize the acids so the pH often rises after cell growth has ceased. The metabolites that are found during secondary metabolism are diverse, and many of them have commercial or therapeutic importance. They include antibiotics, enzymes (e.g. amylases that digest starch and proteolytic enzymes used in biological washing powders), toxins (responsible for many of the 14
symptoms of infection but some also of therapeutic value, e.g. botox — the toxin of Clostridium botulinum) and carbohydrates (e.g. dextran used as a plasma expander and for molecular separations by gel filtration).
4 Microbial cultivation The vast majority of microorganisms of interest in pharmacy and medicine can be cultivated in the laboratory and most of them require relatively simple techniques and facilities. Some organisms are parasites and so can only be grown inside the cells of a host species — which often necessitates mammalian cell culture facilities — and there are a few (e.g. the organism responsible for leprosy) that have never been cultivated outside the living animal. 4.1 Culture media A significant number of common microorganisms are capable of synthesizing all the materials they need for growth (e.g. amino acids, nucleotides and vitamins) from simple carbon and nitrogen sources and mineral salts. Such organisms can grow on truly synthetic (chemically defined) media, but many organisms do not have this capability and need a medium that already contains these biochemicals. Such media are far more commonly used than synthetic ones, and several terms have been used to describe them, e.g. routine laboratory media, general purpose media and complex media. They are complex in the sense that their precise chemical composition is unknown and is likely to vary slightly from batch to batch. In general, they are aqueous solutions of animal or plant extracts that contain hydrolysed proteins, B-group vitamins and carbohydrates. Readily available and relatively inexpensive sources of protein include meat extracts (from those parts of animal carcasses that are not used for human or domestic animal consumption), milk and soya. The protein is hydrolysed to varying degrees to give peptones (by definition not coagulable by heat or ammonium sulphate) or amino acids. Trypsin or other proteolytic enzymes are preferred to acids as a means of hydrolysis because acids
Fundamental features of microbiology
cause more amino acid destruction; the term ‘tryptic’ denotes the use of the enzyme. Many microorganisms require B-group vitamins (but not the other water- or fat-soluble vitamins required by mammals) and this requirement is satisfied by yeast extract. Carbohydrates are used in the form of starch or sugars, but glucose (dextrose) is the only sugar regularly employed as a nutrient. Microorganisms differ in terms of their ability to ferment various sugars and their fermentation patterns may be used as an aid in identification. Thus, other sugars included in culture media are normally present for these diagnostic purposes rather than as carbon and energy sources. Sodium chloride may be incorporated in culture media to adjust osmotic pressure, and occasionally buffers are added to neutralize acids that result from sugar metabolism. Routine culture media may be enriched by the addition of materials like milk, blood or serum, and organisms that need such supplements in order to grow are described as ‘exacting’ in their nutritional requirements. Culture media may be either liquid or solid; the latter term describes liquid media that have been gelled by the addition of agar, which is a carbohydrate extracted from certain seaweeds. Agar at a concentration of about 1–1.5% w/v will provide a firm gel that cannot be liquefied by the enzymes normally produced during bacterial growth (which is one reason it is used in preference to gelatin). Agar is unusual in that the melting and setting temperatures for its gels are quite dissimilar. Fluid agar solutions set at approximately 40°C, but do not reliquefy on heating until the temperature is in excess of 90°C. Thus agar forms a firm gel at 37°C which is the normal incubation temperature for many pathogenic organisms (whereas gelatin does not) and when used as a liquid at 45°C is at a sufficiently low temperature to avoid killing microorganisms — this property is important in pour plate counting methods (see section 5). In contrast to medium ingredients designed to support microbial growth, there are many materials commonly added to selective or diagnostic media whose function is to restrict the growth of certain types of microorganism while permitting or enhancing the growth of others. Examples include antibacterial antibiotics added to fungal media to
suppress bacterial contaminants, and bile to suppress organisms from anatomical sites other than the gastrointestinal tract. Many such additives are used in media for organism identification purposes, and these are considered further in subsequent chapters. The term enrichment sometimes causes confusion in this context. It is occasionally used in the sense of making a medium nutritionally richer to achieve more rapid or profuse growth. Alternatively, and more commonly, an enrichment medium is one designed to permit a particular type of organism to grow while restricting others, so the one that grows increases in relative numbers and is ‘enriched’ in a mixed culture. Solid media designed for the growth of anaerobic organisms usually contain non-toxic reducing agents, e.g. sodium thioglycollate or sulphur-containing amino acids; these compounds create redox potentials of -200 mV or less and so diminish or eliminate the inhibitory effects of oxygen or oxidizing molecules on anaerobic growth. The inclusion of such compounds is less important in liquid media where a sufficiently low redox potential may be achieved simply by boiling; this expels dissolved oxygen, which in unstirred liquids, only slowly resaturates the upper few millimetres of liquid. Redox indicators like methylene blue or resazurin may be incorporated in anaerobic media to confirm that a sufficiently low redox potential has been achieved. Media for yeasts and moulds often have a lower pH (5.5–6.0) than bacterial culture media (7.0–7.4). Lactic acid may be used to impart a low pH because it is not, itself, inhibitory to fungi at the concentrations used. Some fungal media that are intended for use with specimens that may also contain bacteria may be supplemented with antibacterial antibiotics, e.g. chloramphenicol or tetracyclines. 4.2 Cultivation methods Most bacteria and some yeasts divide by a process of binary fission whereby the cell enlarges or elongates, then forms a cross-wall (septum) that separates the cell into two more-or-less equal compartments each containing a copy of the genetic material. Septum formation is often followed by constriction such that the connection between the two cell compartments is progressively reduced (see 15
Chapter 2
Fig. 2.1a) until finally it is broken and the daughter cells separate. In bacteria this pattern of division may take place every 25–30 minutes under optimal conditions of laboratory cultivation, although growth at infection sites in the body is normally much slower owing to the effects of the immune system and scarcity of essential nutrients, particularly iron. Growth continues until one or more nutrients is exhausted, or toxic metabolites (often organic acids) accumulate and inhibit enzyme systems. Starting from a single cell many bacteria can achieve concentrations of the order of 109 cells ml-1 or more following overnight incubation in common liquid media. At concentrations below about 107 cells ml-1 culture media are clear, but the liquid becomes progressively more cloudy (turbid) as the concentration increases above this value; turbidity is, therefore, an indirect means of monitoring culture growth. Some bacteria produce chains of cells, and some elongated cells (filaments) that may exhibit branching to produce a tangled mass resembling a mould mycelium (Fig. 2.1d). Many yeasts divide by budding (see section 1.2.3 and Fig. 2.1b) but they, too, would normally grow in liquid media to produce a turbid culture. Moulds, however, grow by extension and branching of hyphae to produce a mycelium (Fig. 2.1c) or, in agitated liquid cultures, pellet growth may arise. When growing on solid media in Petri dishes (often referred to as ‘plates’) individual bacterial cells can give rise to colonies following overnight incubation under optimal conditions. A colony is simply a collection of cells arising by multiplication of a single original cell or a small cluster of them (called a colony-forming unit or CFU). The term ‘colony’ does not, strictly speaking, imply any particular number of cells, but it is usually taken to mean a number sufficiently large to be visible by eye. Thus, macroscopic bacterial colonies usually comprise hundreds of thousands, millions or tens of millions of cells in an area on a Petri dish that is typically 1–10 mm in diameter (Fig. 2.1e). Colony size is limited by nutrient availability and/or waste product accumulation in just the same way as cell concentration in liquid media. Colonies vary between bacterial species, and their shapes, sizes, opacities, surface markings and pigmentation may all be characteristic of the species in question, so these 16
properties may be an aid in identification procedures (see Chapter 3). Anaerobic organisms may be grown on Petri dishes provided that they are incubated in an anaerobic jar. Such jars are usually made of rigid plastic with airtight lids, and Petri dishes are placed in them together with a low temperature catalyst. The catalyst, consisting of palladium-coated pellets or wire, causes the oxygen inside the jar to be combined with hydrogen that is generated by the addition of water to sodium borohydride; this is usually contained in a foil sachet that is also placed in the jar. As the oxygen is removed, an anaerobic atmosphere is achieved and this is monitored by an oxidationreduction (redox) indicator; resazurin is frequently used, as a solution soaking a fabric strip. Yeast colonies often look similar to those of bacteria, although they may be larger and more frequently coloured. The appearance of moulds growing on solid microbiological media is similar to their appearance when growing on common foods. The mould colony consists of a mycelium that may be loosely or densely entangled depending on the species, often with the central area (the oldest, most mature region of the colony) showing pigmentation associated with spore production (Fig. 2.1f). The periphery of the colony is that part which is actively growing and it is usually non-pigmented. 4.3 Planktonic and sessile growth Bacteria growing in liquid culture in the laboratory usually exist as individual cells or small aggregates of cells suspended in the culture medium; the term planktonic is used to describe such freely suspended cells. In recent years, however, it has become recognized that planktonic growth is not the normal situation for bacteria growing in their natural habitats. In fact, bacteria in their natural state far more commonly grow attached to a surface which, for many species, may be solid, e.g. soil particles, stone, metal or glass, or for pathogens an epithelial surface in the body, e.g. lung or intestinal mucosa. Bacteria attached to a substrate in this way are described as sessile, and are said to exhibit the biofilm or microcolony mode of growth. Planktonic cells are routinely used for almost all the testing procedures that have been designed to
Fundamental features of microbiology
assess the activity of antimicrobial chemicals and processes, but the recognition that planktonic growth is not the natural state for many organisms prompted investigations of the relative susceptibilities of planktonic- and biofilm-grown cells to antibiotics, disinfectants and decontamination or sterilization procedures. In many cases it has been found that planktonic and sessile bacteria exhibit markedly different susceptibilities to these lethal agents, and this has prompted a reappraisal of the appropriateness of some of the procedures used (see Chapters 11 and 13).
5 Enumeration of microorganisms In a pharmaceutical context there are several situations where it is necessary to measure the number of microbial cells in a culture, sample or specimen: • when measuring the levels of microbial contamination in a raw material or manufactured medicine • when evaluating the effects of an antimicrobial chemical or decontamination process • when using microorganisms in the manufacture of therapeutic agents • when assessing the nutrient capability of a growth medium. In some cases it is necessary to know the total number of microbial cells present, i.e. both living and dead, e.g. in vaccine manufacture dead and
living cells may both produce an immune response, and in pyrogen testing both dead and living cells induce fever when injected into the body. However, in many cases it is the number or concentration of living cells that is required. The terminology in microbial counting sometimes causes confusion. A total count is a counting procedure enumerating both living and dead cells, whereas a viable count, which is far more common, records the living cells alone. However, the term total viable count (TVC) is used in most pharmacopoeias and by many regulatory agencies to mean a viable count that records all the different species or types of microorganism that might be present in a sample. Table 2.2 lists the more common counting methods available. The first three traditional methods of viable counting all operate on the basis that a living cell (or a small aggregate or ‘clump’ of cells) will give rise to a visible colony when introduced into or onto the surface of a suitable medium and incubated. Thus, the procedure for pour plating usually involves the addition of a small volume (typically 1.0 ml) of sample (or a suitable dilution thereof) into molten agar at 45°C which is then poured into empty sterile Petri dishes. After incubation the resultant colonies are counted and the total is multiplied by the dilution factor (if any) to give the concentration in the original sample. In a surface spread technique the sample (usually 0.1–0.25 ml) is spread over the surface of agar which has
Table 2.2 Traditional and rapid methods of enumerating cells Traditional methods Viable counts
Total counts
Rapid methods (Indirect viable counts)
1 Pour plate (counting colonies in agar) 2 Surface spread or surface drop (Miles Misra) methods (counting colonies on agar surface) 3 Membrane filter methods (colonies growing on membranes on agar surface) 4 Most probable number (counts based on the proportion of liquid cultures growing after receiving low inocula)
1 Direct microscopic counting (using Helber or haemocytometer counting chambers) 2 Turbidity methods (measures turbidity (opacity) in suspensions or cultures) 3 Dry weight determinations 4 Nitrogen, protein or nucleic acid determinations
1 Epifluorescence (uses dyes that give characteristic fluorescence only in living cells) often coupled to image analysis 2 Adenosine triphosphate (ATP) methods (measures ATP production in living cells using bioluminescence) 3 Impedance (measures changes in resistance, capacitance or impedance in growing cultures) 4 Manometric methods (measure oxygen consumption or CO2 production by growing cultures)
17
Chapter 2
previously been dried to permit absorption of the added liquid. The Miles Misra (surface drop method) is similar in principle, but several individual drops of culture are allowed to spread over discrete areas of about 1 cm diameter on the agar surface. These procedures are suitable for samples that are expected to contain concentrations in excess of approximately 100 CFU ml-1 so that the number of colonies arising on the plate is sufficiently large to be statistically reliable. If there are no clear indications of the order of magnitude of the concentration in the sample, it is necessary to plate out the sample at each of two, three or more (decimal, i.e. 10-fold) dilutions so as to obtain Petri dishes with conveniently countable numbers of colonies (usually taken to be 30–300 colonies). If 30 is accepted as the lowest reliable number to count and a pour plate method uses a 1.0-ml sample, it follows that the procedures described above are unsuitable for any sample that is expected to contain 200 mm in diameter. The small size of bacteria has a number of implications with regard to their biological properties, most notably increased and more efficient transport rates. This advantage allows bacteria far more rapid growth rates than eukaryotic cells. While the classification of bacteria is immensely complex, nowadays relying very much on 16S ribosomal DNA sequencing data, a more simplistic approach is to divide them into major groups on purely morphological grounds. The majority of bacteria are unicellular and possess simple shapes, e.g. round (cocci), cylindrical (rod) or ovoid. Some rods are curved (vibrios), while longer rigid curved organisms with multiple spirals are known as spirochaetes. Rarer morphological forms include the actinomycetes which are rigid bacteria resembling fungi that may grow as lengthy branched filaments; the mycoplasmas which lack a conventional peptidoglycan (murein) cell wall and are highly pleomorphic organisms of indefinite shape; and some miscellaneous bacteria comprising stalked, sheathed, budded and slime-producing forms often associated with aquatic and soil environments.
Bacteria
Capsule
Fimbriae Ribosomes
[NAM Pili
Slime
Mesosome Cytoplasm
Flagella Membrane Plasmid Nucleoid Cell Wall
NAG
NAM
L-ala
L-ala
D-glu
D-glu
Meso-DAP
Meso-DAP
D-ala
D-ala
NAG] interbridge
D-ala DAP D-glu
Fig. 3.1 Diagram of a bacterial cell. Features represented
L-ala
above the dotted line are only found in some bacteria, whereas those below the line are common to all bacteria.
NAG
NAM
NAG
Fig. 3.2 Structure of Escherichia coli peptidoglycan.
Often bacteria remain together in specific arrangements after cell division. These arrangements are usually characteristic of different organisms and can be used as part of a preliminary identification. Examples of such cellular arrangements include chains of rods or cocci, paired cells (diplococci), tetrads and clusters. 2.2 Cellular components Compared with eukaryotic cells, bacteria possess a fairly simple base cell structure, comprising cell wall, cytoplasmic membrane, nucleoid, ribosomes and occasionally inclusion granules (Fig. 3.1). Nevertheless it is important for several reasons to have a good knowledge of these structures and their functions. First, the study of bacteria provides an excellent route for probing the nature of biological processes, many of which are shared by multicellular organisms. Secondly, at an applied level, normal bacterial processes can be customized to benefit society on a mass scale. Here, an obvious example is the large-scale industrial production (fermentation) of antibiotics. Thirdly, from a pharmaceutical and health-care perspective, it is important to be able to know how to kill bacterial contaminants and disease-causing organisms. To treat infections antimicrobial agents are used to inhibit the growth of bacteria, a process known as antimicrobial chemotherapy. The essence of antimicrobial chemotherapy is selective toxicity (Chapters 10, 12 and 14), which is achieved by exploiting differences between the structure and metabolism of bacteria and host cells. Selective toxicity is, therefore, most efficient when a similar target does not exist in the
host. Examples of such targets will be noted in the following sections. 2.2.1 Cell wall The bacterial cell wall is an extremely important structure, being essential for the maintenance of the shape and integrity of the bacterial cell. It is also chemically unlike any structure present in eukaryotic cells and is therefore an obvious target for antibiotics that can attack and kill bacteria without harm to the host (Chapter 12). The primary function of the cell wall is to provide a strong, rigid structural component that can withstand the osmotic pressures caused by high chemical concentrations of inorganic ions in the cell. Most bacterial cell walls have in common a unique structural component called peptidoglycan (also called murein or glycopeptide); exceptions include the mycoplasmas, extreme halophiles and the archaea. Peptidoglycan is a large macromolecule containing glycan (polysaccharide) chains that are cross-linked by short peptide bridges. The glycan chain acts as a backbone to peptidoglycan, and is composed of alternating residues of N-acetyl muramic acid (NAM) and N-acetyl glucosamine (NAG). To each molecule of NAM is attached a tetrapeptide consisting of the amino acids lalanine, d-alanine, d-glutamic acid and either lysine or diaminopimelic acid (DAP). This glycan tetrapeptide repeat unit is cross-linked to adjacent glycan chains, either through a direct peptide linkage or a peptide interbridge (Fig. 3.2). The types and numbers of cross-linking amino acids vary from organism to organism. Other unusual features of the 25
Chapter 3 Surface protein
Teichoic acid
Lipoteichoic acid
Peptidoglycan
Cytoplasmic membrane
Fig. 3.3 Structure of the Gram-positive
cell wall.
cell wall that provide potential antimicrobial targets are DAP and the presence of two amino acids that have the d-configuration. Bacteria can be divided into two large groups, Gram-positive and Gram-negative, on the basis of a differential staining technique called the Gram stain. Essentially, the Gram stain consists of treating a film of bacteria dried on a microscope slide with a solution of crystal violet, followed by a solution of iodine; these are then washed with an alcohol solution. In Gram-negative organisms the cells lose the crystal violet–iodine complex and are rendered colourless, whereas Gram-positive cells retain the dye. Regardless, both cell types are counter-stained with a different coloured dye, e.g. carbol fuchsin, which is red. Hence, under the light microscope Gram-negative cells appear red while Gram-positive cells are purple. These marked differences in response reflect differences in cell wall structure. The Gram-positive cell wall consists primarily of a single type of molecule whereas the Gram-negative cell wall is a multilayered structure and quite complex. The cell walls of Gram-positive bacteria are quite thick (20–80 nm) and consist of between 60% and 80% peptidoglycan, which is extensively crosslinked in three dimensions to form a thick polymeric mesh (Fig. 3.3). Gram-positive walls frequently contain acidic polysaccharides called teichoic acids; these are either ribitol phosphate or glycerol phosphate molecules that are connected by phos26
phodiester bridges. Because they are negatively charged, teichoic acids are partially responsible for the negative charge of the cell surface as a whole. Their function may be to effect passage of metal cations through the cell wall. In some Gram-positive bacteria glycerol–teichoic acids are bound to membrane lipids and are termed lipoteichoic acids. During an infection, lipoteichoic acid molecules released by killed bacteria trigger an inflammatory response. Cell wall proteins, if present, are generally found on the outer surface of the peptidoglycan. The wall, or more correctly, envelope of Gramnegative cells is a far more complicated structure (Fig. 3.4). Although they contain less peptidoglycan (10–20% of wall), a second membrane structure is found outside the peptidoglycan layer. This outer membrane is asymmetrical, composed of proteins, lipoproteins, phospholipids and a component unique to Gram-negative bacteria, lipopolysaccharide (LPS). Essentially, the outer membrane is attached to the peptidoglycan by a lipoprotein, one end of which is covalently attached to peptidoglycan and the other end is embedded in the outer membrane. The outer membrane is not a phospholipid bilayer although it does contain phospholipids in the inner leaf, and its outer layer is composed of LPS, a polysaccharide–lipid molecule. Proteins are also found in the outer membrane, some of which form trimers and traverse the whole membrane and in so doing form water-filled channels or porins through which small molecules can pass. Other
Bacteria Lipopolysaccharide
Receptor protein Porin
Lipoprotein
Outer membrane Periplasmic protein Periplasm Fig. 3.4 Structure of the Gram-negative
cell envelope.
Peptidoglycan
Fig. 3.5 Schematic representation of
lipopolysaccharide (LPS).
Lipid A
proteins are found at either the inner or outer face of the membrane. The LPS (Fig. 3.5) is an important molecule because it determines the antigenicity of the Gramnegative cell and it is extremely toxic to animal cells. The molecule consists of three regions, namely lipid A, core polysaccharide and O-specific polysaccharide. The lipid A portion is composed of a disaccharide of glucosamine phosphate bound to fatty acids and forms the outer leaflet of the membrane. It is the lipid A component that is responsible for the toxic and pyrogenic properties of Gram-negative bacteria. Lipid A is linked to the core polysaccharide by the unique molecule ketodeoxyoctonate (KDO), and at the other end of the core is the O-polysaccharide (O-antigen), which usually contains six-carbon sugars as well as one or more unusual deoxy sugars such as abequose. Although the outer membrane is relatively permeable to small molecules, it is not permeable to enzymes or large molecules. Indeed, one of the major functions of the outer membrane may be to keep
KDO
Core
O-antigen
certain enzymes that are present outside the cytoplasmic membrane from diffusing away from the cell. Moreover, the outer membrane is not readily penetrated by hydrophobic compounds and is, therefore, resistant to dissolution by detergents. The region between the outer surface of the cytoplasmic membrane and the inner surface of the outer membrane is called the periplasm. This occupies a distance of about 12–15 nm, is gel-like in consistency and, in addition to the peptidoglycan, contains sugars and an abundance of proteins including hydrolytic enzymes and transport proteins. Table 3.2 summarizes the major differences in wall composition between Gram-positive and Gram-negative cells. 2.2.2 Cytoplasmic membrane Biochemically, the cytoplasmic membrane is a fragile, phospholipid bilayer with proteins distributed randomly throughout. These are involved in the various transport and enzyme functions 27
Chapter 3 Table 3.2 Gram-positive and Gram-negative cell wall composition
Feature
Gram-positive cells
Gram-negative cells
Peptidoglycan Teichoic acid Lipoteichoic acid Lipoprotein Lipopolysaccharide Protein Lipid
60–80% Present Present Absent Absent c. 15% c. 2%
10–20% Absent Absent Present Present c. 60% c. 20%
associated with the membrane. A major difference in chemical composition between prokaryotic and eukaryotic cells is that eukaryotes have sterols in their membranes (e.g. cholesterol) whereas prokaryotes do not. The cytoplasmic membrane serves many functions, including transport of nutrients, energy generation and electron transport; it is the location for regulatory proteins and biosynthetic proteins, and it acts as a semi-permeable selectivity barrier between the cytoplasm and the cell environment. Invaginations of the cytoplasmic membrane are referred to as mesosomes. Those that form near the septum of Gram-positive cells serve as organs of attachment for the bacterial chromosome. 2.2.3 Cytoplasm The cytoplasm consists of approximately 80% water and contains enzymes that generate ATP directly by oxidizing glucose and other carbon sources. The cytoplasm also contains some of the enzymes involved in the synthesis of peptidoglycan subunits. Ribosomes, the DNA genome (nucleoid) and inclusion granules are also found in the cytoplasm. 2.2.4 Nucleoid The bacterial chromosome exists as a singular, covalently closed circular molecule of doublestranded DNA comprising approximately 4600 kilobase pairs. It is complexed with small amounts of proteins and RNA, but unlike eukaryotic DNA, 28
is not associated with histones. The DNA, if linearized, would be about 1 mm in length. In order to package this amount of material the cell requires that the DNA is supercoiled into a number of domains (c. 50) and that the domains are associated with each other and stabilized by specific proteins into an aggregated mass or nucleoid. The enzymes, topoisomerases, that control topological changes in DNA architecture are different from their eukaryotic counterparts (which act on linear chromosomes) and therefore provide a unique biochemical target for antibiotic action. 2.2.5 Plasmids Plasmids are relatively small, circular pieces of double-stranded extrachromosomal DNA. They are capable of autonomous replication and encode for many auxiliary functions that are not usually necessary for bacterial growth. One such function of great significance is that of antibiotic resistance (Chapter 13). Plasmids may also transfer readily from one organism to another, and between species, thereby increasing the spread of resistance. 2.2.6 Ribosomes The cytoplasm is densely packed with ribosomes. Unlike eukaryotic cells these are not associated with a membranous structure; the endoplasmic reticulum is not a component of prokaryotic cells. Bacterial ribosomes are 70S in size, comprising two subunits of 30S and 50S. This is smaller than eukaryotic ribosomes, which are 80S in size (40S and 60S subunits). Differences will therefore exist in the size and geometry of RNA binding sites. 2.2.7 Inclusion granules Bacteria occasionally contain inclusion granules within their cytoplasm. These consist of storage material composed of carbon, nitrogen, sulphur or phosphorus and are formed when these materials are replete in the environment to act as repositories of these nutrients when limitations occur. Examples include poly-b-hydroxybutyrate, glycogen and polyphosphate.
Bacteria
2.3 Cell surface components
2.3.3 Pili
The surface of the bacterial cell is the portion of the organism that interacts with the external environment most directly. As a consequence, many bacteria deploy components on their surfaces in a variety of ways that allow them to withstand and survive fluctuations in the growth environment. The following sections describe a few of these components that are commonly found, although not universally, that allow bacteria to move, sense their environment, attach to surfaces and provide protection from harsh conditions.
Pili are morphologically and chemically similar to fimbriae, but they are present in much smaller numbers (< 10) and are usually longer. They are involved in the genetic exchange process of conjugation (section 6.3).
2.3.1 Flagella Bacterial motility is commonly provided by flagella, long (c. 12 mm) helical-shaped structures that project from the surface of the cell. The filament of the flagellum is built up from multiple copies of the protein flagellin. Where the filament enters the surface of the bacterium, there is a hook in the flagellum, which is attached to the cell surface by a series of complex proteins called the flagellar motor. This rotates the flagellum, causing the bacterium to move through the environment. The numbers and distribution of flagella vary with bacterial species. Some have a single, polar flagellum, whereas others are flagellate over their entire surface (peritrichous); intermediate forms also exist.
2.3.4 Capsules and slime layers Many bacteria secrete extracellular polysaccharides (EPS) that are associated with the exterior of the bacterial cell. The EPS is composed primarily of c. 2% carbohydrate and 98% water, and provides a gummy exterior to the cell. Morphologically, two extreme forms exist: capsules, which form a tight, fairly rigid layer closely associated with the cell, and slimes, which are loosely associated with the cell. Both forms function similarly, to offer protection against desiccation, to provide a protective barrier against the penetration of biocides, disinfectants and positively charged antibiotics, to protect against engulfment by phagocytes and protozoa and to act as a cement binding cells to each other and to the substratum in biofilms. One such polymer that performs all these functions is alginate, produced by Pseudomonas aeruginosa. Bacterial EPS such as alginate and the high molecular weight dextrans produced by Leuconostoc mesenteroides may be harvested and used as pharmaceutical aids, surgical dressings and drug delivery systems.
2.3.2 Fimbriae Fimbriae are structurally similar to flagella, but are not involved in motility. While they are straighter, more numerous and considerably thinner and shorter (3 mm) than flagella, they do consist of protein and project from the cell surface. There is strong evidence to suggest that fimbriae act primarily as adhesins, allowing organisms to attach to surfaces, including animal tissues in the case of some pathogenic bacteria, and to initiate biofilm formation. Fimbriae are also responsible for haemagglutination and cell clumping in bacteria. Among the best characterized fimbriae are the type I fimbriae of enteric (intestinal) bacteria.
2.3.5 S-layers Many bacteria produce a cell surface layer composed of a two-dimensional paracrystalline array of proteins termed S-layers. These show various symmetries and can associate with a variety of cell wall structures. Although their true function is currently unknown it is likely that they will act to a certain extent as an external permeability barrier.
3 Biofilms Any surface, whether it is animate or inanimate, is of considerable importance as a microbial habitat owing to the adsorption of nutrients. As such, a 29
Chapter 3
nutrient-rich micro-environment is produced in a nutrient-poor macro-environment whenever a surface–liquid interface exists. Consequently, microbial numbers and activity are usually much greater on a surface than in suspension. Hence, in many natural, medical and industrial settings bacteria attach to surfaces and form multilayered communities called biofilms. These commonly contain more than one species of bacteria, which cooperatively exist together as a functional, dynamic consortium. Biofilm formation usually begins with pioneer cells attaching to a surface, either through the use of specific adhesins such as fimbriae, or nonspecifically by EPS. Once established, these cells grow and divide to produce microcolonies, which with time, eventually coalesce to produce a biofilm. A key characteristic of biofilms is the enveloping of the attached cells in EPS. Biofilms help trap nutrients for growth of the enclosed cells and help prevent detachment of cells on surfaces in flowing systems. Biofilms have a number of significant implications in medicine and industry. In the human body the resident cells within the biofilm are not exposed to attack by the immune system and in some instances can exacerbate the inflammatory response. An example of this type is shown by the growth of P. aeruginosa as an alginate-enclosed biofilm in the lungs of cystic fibrosis patients. Bacterial biofilms are also profoundly less susceptible to antimicrobial agents than their free-living, planktonic counterparts. As a consequence, bacterial biofilms that form on contaminated medical implants and prosthetic devices, manufacturing surfaces or fluid conduit systems are virtually impossible to eliminate with antibiotics or biocides. In these situations antimicrobial resistance occurs as a population or community response.
4 Bacterial sporulation In a few bacterial genera, notably Bacillus and Clostridium, a unique process takes place in which the vegetative cell undergoes a profound biochemical change to give rise to a specialized structure called an endospore or spore (Fig. 3.6). This process of sporulation is not part of a reproductive cycle, 30
sporulation
1 X vegetative cell
1 X endospore
germination Fig. 3.6 Bacterial sporulation and germination.
but the spore is a highly resistant cell that enables the producing organism to survive in adverse environmental conditions such as lack of moisture or essential nutrients, toxic chemicals, radiation and high temperatures. Because of their extreme resistance to radiation, ethylene oxide and heat, all sterilization processes for pharmaceutical products have been designed to destroy the bacterial spore (Chapter 20). Removal of the environmental stress may lead to germination of the spore back to the vegetative cell form. 4.1 Endospore structure Endospores are differentiated cells that possess a grossly different structure to that of the parent vegetative cell in which they are formed. The structure of the spore is much more complex than that of the vegetative cell in that it has many layers surrounding a central core (Fig. 3.7). The outermost layer is the exosporium composed of protein; within this are the spore coats, which are also proteinaceous but with a high cysteine content, the cortex that consists of loosely cross-linked peptidoglycan and the central core that contains the genome. Characteristic of the spore is the presence of dipicolinic acid and high levels of calcium ions which complex together. The core is also partially dehydrated, containing only 10–30% of the water content of the vegetative cells. Dehydration has been shown to increase resistance to both heat and chemicals. In addition, the pH of the core is about 1 unit lower than the cytoplasm of the vegetative cell and contains high levels of core-specific proteins that
Bacteria Spore coats
Core
Exosporium
volving water uptake and synthesis of new RNA, proteins and DNA until eventually, after a matter of minutes, the vegetative cell emerges from the fractured spore coat and begins to divide again.
Spore wall
5 Bacterial toxins
Cortex Fig. 3.7 Diagram of endospore structure.
bind tightly to the DNA and protect it from potential damage. These core-specific proteins also function as an energy source for the outgrowth or germination of a new vegetative cell from the endospore. 4.2 Endospore formation During endospore formation the vegetative cell undergoes a complex series of biochemical events in cellular differentiation, and many genetically directed changes in the cell that underpin the conversion occur in a series of distinct stages. Sporulation requires that the synthesis of some proteins involved in vegetative cell function cease and that specific spore proteins are made. This is accomplished by activation of a variety of spore-specific genes such as spo and ssp. The proteins coded by these genes catalyse a series of events leading ultimately to the production of a dry, metabolically inert but extremely resistant endospore. The whole process can take only a matter of hours to complete under optimal conditions. 4.3 Endospore germination Although endospores can lie dormant for decades, they can revert back to a vegetative cell very rapidly. Activation of the process may occur through removal of the stress-inducer that initiated sporulation. During germination loss of resistance properties occurs along with a loss of calcium dipicolinate and cortex components, and degradation of the core-specific proteins. Outgrowth occurs, in-
Although bacteria are associated with disease, only a few species are disease-producing or pathogenic (Chapter 7). Of greater concern are those organisms that, if presented with the correct set of conditions, can cause disease, i.e. opportunist pathogens. Examples include Staphylococcus epidermidis, a beneficial organism when present on the skin (its normal habitat) yet potentially fatal if attached to a synthetic heart valve, and P. aeruginosa, a nonpathogenic environmental organism but again potentially lethal in immunocompromised patients. The pathogens cause host damage in a number of ways. In most cases pathogens produce a variety of molecules or factors that promote pathogenesis, among which are the toxins: products of bacteria that produce immediate host cell damage. Toxins have been classified as either endotoxin, i.e. cell wall-related, or exotoxin, products released extracellularly as the organism grows. Endotoxin is the lipid A component of LPS (see section 2.2.1). It possesses multiple biological properties including the ability to induce fever, initiate the complement and blood cascades, activate b lymphocytes and stimulate production of tumour necrosis factor. Endotoxin is generally released from lysed or damaged cells. Care must be taken to eliminate or exclude such heat-resistant material from parenteral products and their delivery systems through a process known as depyrogenation (Chapter 20). Most exotoxins fall into one of three categories on the basis of their structure and activities. These are the A-B toxins, the cytolytic toxins and the superantigen toxins. The A-B toxins consist of a B subunit that binds to a host cell receptor, covalently bound to the A subunit that mediates the enzymic activity responsible for toxicity. Most exotoxins (e.g. diphtheria toxin, cholera toxin) are of the A-B category. The cytolytic toxins such as haemolysins and phospholipases do not have separable A and B 31
Chapter 3
portions but work by enzymically attacking cell constituents, causing lysis. The superantigens also lack an A-B type structure and act by stimulating large numbers of immune response cells to release cytokines, resulting in a massive inflammatory reaction. An example of this type of reaction is Staphylococcus aureus-mediated toxic shock syndrome.
6 Bacterial reproduction and growth kinetics 6.1 Multiplication and division cycle The majority of bacterial cells multiply in number by a process of binary fission. That is, each individual will increase in size until it is large enough to divide into two identical daughter cells. At the point of separation each daughter cell must be capable of growth and reproduction. While each daughter cell will automatically contain those materials that are dispersed throughout the mother cell (mRNA, rRNA, ribosomes, enzymes, cytochromes, etc.), each must also carry at least one copy of the chromosome. The bacterial chromosome is circular and attached to the cytoplasmic membrane where it is able to uncoil during DNA replication. The process of DNA replication proceeds at a fixed rate dependent upon temperature, therefore the time taken to copy an entire chromosome depends on the number of base pairs within it and the growth temperature. For Escherichia coli growing at 37°C replication of the chromosome will take approximately 45 minutes. These copies of the chromosome must then segregate to opposite sides of the cell before cell division can proceed. Division occurs in different ways for Gram-positive and Gram-negative bacteria. Gram-negative cells do not have a rigid cell wall and divide by a process of constriction followed by membrane fusion. Gram-positive cells, on the other hand, having a rigid cell wall, must develop a crosswall (see Fig. 2.1a) that divides the cell into two equal halves. Constriction and cross-wall formation takes approximately 15 minutes to complete. DNA replication, chromosome segregation (Cphase) and cell division (D-phase) occur sequentially in slow-growing cells with generation times of 32
greater than 1 hour and are the final events of the bacterial cell cycle. Cells are able to replicate faster than once every hour by initiating several rounds of DNA replication at a time. Thus partially replicated chromosomes become segregated into the newly formed daughter cells. In this fashion it is possible for some organisms growing under their optimal conditions to divide every 15–20 minutes. Rodshaped organisms maintain their diameter during the cell cycle and increase their mass and volume by a process of elongation. When the length of the cell has approximately doubled then the division/ constriction occurs centrally. Coccal forms increase in size by radial expansion with the division plane going towards the geometric centre. In some genera the successive division planes are always parallel. Under such circumstances the cells appear to form chains (i.e. streptococci). In staphylococci successive division planes are randomized, giving dividing clusters of cells the appearance of a bunch of grapes. Certain genera, e.g. Sarcina, rotate successive division planes by 90° to form tetrads and cubical octets. The appearance of dividing cells under the microscope can therefore be a useful initial guide to identification. 6.2 Population growth When placed in favourable conditions populations of bacteria can increase at remarkable rates, given that each division gives rise to two identical daughter cells, then each has the potential to divide again. Thus cell numbers will increase exponentially as a function of time. For a microorganism growing with a generation time of 20 minutes, one cell will have divided three times within an hour to give a total of eight cells. After 20 hours of continued division at this rate then the accumulated mass of bacterial cells would be approximately 70 kg (the weight of an average man). Ten hours later the mass would be equivalent to the combined body weight of the entire population of the UK. Clearly this does not happen in nature, rather the supply of nutrients becomes exhausted and the organisms grow considerably more slowly, if at all. The time interval between one cell division and the next is called the generation time. When considering a growing culture containing thousands of
Bacteria
cells, a mean generation time is usually calculated. As one cell doubles to become two cells, which then multiply to become four cells and so on, the number of bacteria n in any generation can be expressed as: 1st generation n = 1 ¥ 2 = 21 2nd generation n = 1 ¥ 2 ¥ 2 = 22 3rd generation n = 1 ¥ 2 ¥ 2 ¥ 2 = 23 xth generation n = 1 ¥ 2 x = 2 x For an initial population of No cells, as distinct from one cell, at the xth generation the cell population will be: N = No ¥ 2 x where N is the final cell number, No the initial cell number and x the number of generations. To express this equation in terms of x, then: Log N = log No + x log 2 Log N - log No = x log 2 x=
log N - log No log N - log No = log 2 0.301
x = 3.3(log N - log No ) The actual generation time is calculated by dividing x into t where t represents the hours or minutes of exponential growth. 6.2.1 Growth on solid surfaces If microorganisms are immobilized on a solid surface from which they can derive nutrients and remain moist, cell division will cause the daughter cells to form a localized colony. In spite of the small size of the individual organisms, colonies are easily visible to the naked eye. Indeed microbial growth can often be seen on the tonsils of an infected individual or as colonies on discarded or badly stored foods. In the laboratory solidified growth media are deployed to separate different types of bacteria and also as an aid to enumerating viable cell numbers. These media comprise a nutrient soup (broth) that has been solidified by the addition of agar (see Chapter 2). Agar melts and dissolves in boiling water but will not resolidify until the temperature is below 45°C. Agar media are used in the laboratory either poured as a thin layer into a covered dish
(Petri dish) or contained within a small, capped bottle (slant). If suspensions of different species of bacteria are spread onto the surface of a nutrient agar plate then each individual cell will produce a single visible colony. These may be counted to obtain an estimate of the original number of cells. Different species will produce colonies of slightly different appearance, enabling judgements to be made as to the population diversity. The colour, size, shape and texture of colonies of different species of bacteria vary considerably and form a useful diagnostic aid to identification. Transfer of single colonies from the plate to a slant enables pure cultures of each organism to be maintained, cultured and identified. 6.2.2 Growth in liquids When growing on a solid surface the size of the resultant colony is governed by the local availability of nutrients. These must diffuse through the colony. Eventually growth ceases when the rate of consumption of nutrients exceeds the rate of supply. When grown in liquids the bacteria, being of colloidal dimension and sometimes highly motile, are dispersed evenly through the fluid. Nutrients are therefore equally available to all cells. When considering growth of bacterial populations in liquids it is necessary to consider whether the environment is closed or open with respect to the acquisition of fresh nutrient. Closed systems are typified by batch culture in closed glass flasks. In these waste products of metabolism are retained and all the available nutrients are present at the beginning of growth. Open systems on the other hand have a continual supply of fresh nutrients and removal of waste products. 6.2.2.1 Liquid batch culture (closed) Figure 3.8 shows the pattern of population growth obtained when a small sample of bacteria is placed within a suitable liquid growth medium held in a glass vessel. As the increase in cell numbers is exponential (1, 2, 4, 8, 16, etc.) then during active growth a logarithmic plot of cell number against time gives a straight line (B). This period is often referred to as the logarithmic growth phase, during which the generation or doubling time may be calculated from the slope of the line. However, the 33
Chapter 3
Log growth
A
B
C
D
Time Fig. 3.8 Typical bacterial growth curve in closed batch liquid culture. (A) Lag or adaptive phase; (B) logarithmic or exponential phase; (C) stationary phase; (D) decline phase.
exponential phase is preceded by a lag period (A), during which time the inoculum adapts its physiology to that required for growth on the available nutrients. As growth proceeds nutrients are consumed and waste materials accumulate. This has the effect of reducing the rate of growth (late logarithmic phase) towards an eventual halt (stationary phase, C). Starvation during the stationary phase will eventually lead to the death of some of the cells and adaptation to a dormant state in others (decline phase, D). Patterns of growth such as this occur within inadequately preserved pharmaceutical products, in water storage tanks and in industrial fermentations. 6.2.2.2 Growth in open culture Except under circumstances of feast–famine, growth of bacteria in association with humans and in our environment is subject to a gradual but continuous provision of nutrients and a dilution of waste products. Under such circumstances the rate of growth of bacteria is governed by the rate of supply of nutrients and the population size. Accordingly, bacteria in our gastrointestinal tracts receive a more or less continuous supply of food and excess bacteria are voided with the faeces (indeed bacteria make up > 90% of the dry mass of faeces). In many situations the bacteria become immobilized, as a biofilm, upon a surface and extract nutrients from the bulk fluid phase. 34
6.3 Growth and genetic exchange For many years it was thought that bacteria, dividing by binary fission, had no opportunity for the exchange of genetic material and could only adapt and evolve through mutation of genes. This is not only untrue but masks the profound ability of bacteria to exchange and share DNA across diverse genera. This is of particular significance because it enables bacterial populations to adapt rapidly to changes in their environment, whether this is related to the appearance of a novel food or to the deployment of antibacterial chemicals and antibiotics. Three major processes of genetic exchange can be identified in bacteria — transformation, transduction and conjugation. Further details of these processes appear in Chapter 13 dealing with the development and spread of antibiotic resistance. 6.3.1 Transformation In 1928 Griffith noticed that a culture of Streptococcus pneumoniae that had mutated to become deficient in capsule production could be restored to its normal capsulate form by incubation with a cellfree filtrate taken from a culture of the normal strain. While this discovery preceded the discovery of DNA as the genetic library and was only poorly understood at the time, it demonstrated the ability of certain types of bacteria to absorb small pieces of naked DNA from the environment that may recombine into the recipient chromosome. The process has become known as transformation and is likely to occur naturally in situations such as septic abscesses and in biofilms where high cell densities are associated with death and lysis of significant portions of the population. Transformation is also exploited in molecular biology as a means of transferring genes between different types of bacteria. 6.3.2 Transduction Viruses are discussed more fully elsewhere (Chapter 5); however, there is a group of viruses, called bacteriophages, which have bacterial cells as their hosts. These bacteriophages inject viral DNA into the host
Bacteria
cell. This viral DNA is then replicated and transcribed at the expense of the host and assembled into new viral particles. Under normal circumstances the host cell becomes lysed in order to release the viral progeny, but in exceptional circumstances, rather than enter a replication cycle the viral DNA becomes incorporated, by recombination, into the chromosome of the bacterium. This is known as a temperate phage. The viral DNA thus forms part of the bacterial chromosome and will be copied to all daughter cells. Temperate phage will become active once again at a low frequency and phasing between temperate and lytic forms ensures the long-term survival of the virus. Occasionally during this transition back to the lytic form the excision of the viral DNA from the bacterial chromosome is inaccurate. The resultant virus may then either be defective, if viral DNA has been lost, or it may carry additional DNA of bacterial origin. Subsequent temperate infections caused by the latter virions will result in this bacterial DNA having moved between cells: a process of gene movement known as transduction. As the host range of some bacteriophages is broad then such processes can move DNA between diverse species. 6.3.3 Conjugation Conjugation is thought to have evolved through transduction, and relates to the generation of defective viral DNA. This can be transcribed to produce singular viral elements, which cannot assemble or lyse the host cell. Such DNA strands are known as plasmids. They are circular and can either be integrated into the main chromosome, in which case they are replicated along with the chromosome and passed to daughter cells, or they are separate from it and can replicate independently. The simplest form of plasmid is the F-factor (fertility factor); this can be transcribed at the cell membrane to generate an F-pilus within the cell envelope and cells containing an F-factor are designated F+. The F-pilus is a hollow appendage that is capable of transferring DNA from one cell to another, through a process that is very similar to the injection of viral DNA into a cell during infection. In its simplest form an unassociated F-factor will simply transfer a copy to a recipient cell, and such a transfer process is known as
conjugation. Integration with, and dissociation of, the F-factor with the chromosome occurs randomly. When it is in the integrated form, designated Hfr (high frequency of recombination), then not only can a copy of the plasmid DNA be transferred across the F-pilus but so also can a partial or complete copy of the donor chromosome. Subsequent recombination events incorporate the new DNA into the recipient chromosome. Just as the excision of temperate viral DNA from the host chromosome could be inaccurate, and lead to additions and deletions from the sequence, so too can the F-factor gather chromosomal DNA as the host cells change from Hfr to F+. In such instances the plasmid that is formed will transfer not only itself but also this additional DNA into recipient cells. This is particularly significant because the unassociated plasmid can replicate autonomously from the chromosome to achieve a high copy number. It can also be transferred simultaneously to many recipient bacteria. If the transported DNA encoded a mechanism of antibiotic resistance (Chapter 13) it would not be difficult to imagine how whole populations could rapidly acquire the resistance characteristics.
7 Environmental factors that influence growth and survival The rate of growth of a microbial population depends upon the nature and availability of water and nutrients, temperature, pH, the partial pressure of oxygen and solute concentrations. In many laboratory experiments the microorganisms are provided with an excess of complex organic nutrients and are maintained at optimal pH and temperature. This enables growth to be very rapid and the results visualized within a relatively short time period. Such idealized conditions rarely exist in nature, where microorganisms not only compete with one another for nutrients but also grow under suboptimal conditions. Particular groups of organisms are adapted to survive under particular conditions; thus Gram-negative bacteria tend to be aquatic whereas Gram-positive bacteria tend to prefer more arid conditions such as the skin. The next two sections of this chapter will consider separately the 35
Chapter 3 4 units
Enzyme reactions at maximum rate
30∞C Range
Optimum 7.2–7.6 Growth Growth
Optimum Min Max
Temperature Membrane gelling
Protein denaturation
Fig. 3.9 The effect of temperature on bacterial growth.
physicochemical factors that affect growth and survival of bacteria, and the availability and nature of the available nutrients. 7.1 Physicochemical factors that affect growth and survival of bacteria 7.1.1 Temperature Earlier in this chapter various classes of bacteria (thermophile, mesophile, etc.) were described according to the range of temperatures under which they could grow. The majority of bacteria that have medical or pharmaceutical significance are mesophiles and have optimal growth temperatures between ambient and body temperature (37°C). Individual species of bacteria also have a range of temperatures under which they can actively grow and multiply (permissive temperatures). For every organism there is a minimum temperature below which no growth occurs, an optimum temperature at which growth is most rapid and a maximum temperature above which growth is not possible (Fig. 3.9). As temperatures rise, chemical and enzymic reactions within the cell proceed more rapidly, and growth becomes faster until an optimal rate is achieved. Beyond this temperature certain proteins may become irreversibly damaged through thermal lysis, resulting in a rapid loss of cell viability. The optimum temperature for growth is much nearer the maximum value than the minimum and the range of the permissive temperatures can be quite narrow (3–4°C) for obligate pathogens yet 36
pH Fig. 3.10 The effect of pH on bacterial growth.
broad (10–20°C) for environmental isolates, reflecting the range of temperatures that they are likely to encounter in their specialized niches. If the temperature exceeds the permissive range then provided that lethal temperatures are not achieved (c. 60°C for most Gram-negative mesophiles) the organisms will survive but not grow. Temperatures of 105°C and above are rapidly lethal and can be deployed to sterilize materials and products. Generally bacteria are able to survive temperatures beneath the permissive range provided that they are gradually acclimatized to them. 7.1.2 pH As for temperature, each individual microorganism has an optimal pH for growth and a range about that optimum where growth can occur albeit at a slower pace. Unlike the response to temperature, pH effects on growth are bell-shaped (Fig. 3.10), and extremes of pH can be lethal. Generally those microorganisms that have medical or pharmaceutical significance have pH growth optima of between 7.4 and 7.6 but may grow suboptimally at pH values of 5–8.5. Thus growth of lactobacilli within the vaginal vault reduces the pH to approximately 5.5 and prevents the growth of many opportunist pathogens. Accordingly, the pH of a pharmaceutical preparation may dictate the range of microorganisms that could potentially cause its spoilage.
Bacteria
7.1.3 Water activity/solutes Water is essential for the growth of all known forms of life. Gram-negative bacteria are particularly adapted to an existence in, and are able to extract trace nutrients from, the most dilute environments. This adaptation has its limitations because the Gram-negative cell envelope cannot withstand the high internal osmotic pressures associated with rapid re-hydration after desiccation and the organisms are unable to grow in the presence of high concentrations of solute. The availability of water is reflected in the water activity of a material or liquid. Water activity (Aw) is defined as the vapour pressure of water in the space above the material relative to the vapour pressure above pure water at the same temperature and pressure. Pure water by definition has an Aw of 1.00. Pharmaceutical creams might have Aw values of 0.8–0.98, whereas strawberry jam might have an Aw of c. 0.7. Generally Gramnegative bacteria cannot grow if the Aw is below 0.97, whereas Gram-positive bacteria can grow in materials with Aw of 0.8–0.98 and can survive rehydration after periods of desiccation, hence their dominance in the soil. Yeasts and moulds can grow at low Aw values, hence their appearance on moist bathroom walls and on the surface of jam. The water activity of a pharmaceutical product can markedly affect its vulnerability to spoilage contaminants (see Chapter 16). 7.1.4 Availability of oxygen For many aerobic microorganisms oxygen acts as the terminal electron acceptor in respiration and is essential for growth. Alternate terminal electron acceptors are organic molecules whose reduction leads to the generation of organic acids such as lactic acid. They can sometimes be utilized under conditions of low oxygen or where carbon substrate is in excess (fermentation), and highly specialized groups of microorganisms can utilize inorganic materials such as iron as electron acceptors (e.g. ironsulphur bacteria). Different groups of organisms therefore vary in their dependence upon oxygen. Paradoxically there are many bacteria for which oxygen is highly toxic (obligate anaerobes), so the presence or absence of oxygen within a nutrient en-
vironment can profoundly affect both the rate and nature of the microbial growth obtained. Strongly oxygen-dependent bacteria will tend to grow as a thin pellicle on the surfaces of liquid media where oxygen is most available. Special media and anaerobic chambers are required to grow obligate anaerobes within the laboratory, yet such organisms persist and actively grow within the general environment. This is because the close proximity of strongly aerobic cells and anaerobes will create an anoxic micro-environment in which the anaerobe can flourish. This is particularly the case for the mouth and gastrointestinal tract where obligate anaerobes such as Bacteroides and Fusobacter can be found in association with strongly aerobic streptococci. The inability of oxygen to diffuse adequately into a liquid culture is often the factor that causes an onset of stationary phase, so culture density is limited by oxygen demand. The cell density at stationary phase can often be increased, therefore, by shaking the flask or providing baffles. Diffusion of oxygen may also be a factor limiting the size of bacterial colonies formed on an agar surface. 7.2 Nutrition and growth Bacteria vary considerably in their requirements for nutrients and in their ability to synthesize for themselves various vitamins and growth factors. Clearly the major elemental requirements for growth will match closely the elemental composition of the bacteria themselves. In this fashion there is a need for the provision of carbon, nitrogen, water, phosphorus, potassium and sulphur with a minor requirement for trace elements such as magnesium, calcium, iron, etc. The most independent classes of bacteria are able to derive much of their nutrition from simple inorganic forms of these elements. These organisms are called chemolithotrophs and can even utilize atmospheric carbon dioxide and nitrogen as sources of carbon and nitrogen. Indeed, such bacteria are, in addition to the green plants and algae, a major source of organic molecules and so they are more beneficial than problematic to man. The majority of bacteria require a fixed carbon source, usually in the form of a sugar, but this may also be obtained from complex organic molecules 37
Chapter 3
such as benzene, paraffin waxes and proteins. Nitrogen can generally be obtained from ammonium ions but is also available by deamination of amino acids, which can thus provide both carbon and nitrogen sources simultaneously. Many classes of bacteria are auxotrophic and can grow on simple sugars together with ammonium ions, a source of potassium and trace elements. Such bacteria can synthesize for themselves all the amino acids and ancillary factors required for growth and division. These bacteria, e.g. pseudomonads and Achromobacter species, are generally free-living environmental strains but they can sometimes cause infections in immunocompromised people. In the laboratory they can be grown in simple salts media with few, if any, complex supplements. The rate of growth of such organisms depends not only on temperature and pH but also on the nature of the carbon and nitrogen sources. Thus, a faster rate of growth is often obtained when glucose or succinate is the carbon source rather than lactose or glycerol, and when amino acids are provided as sources of nitrogen rather than ammonium salts. If faced with a choice of carbon and nitrogen sources then the bacteria will adapt their physiology to the preferred substrate and only when this is depleted will they turn their attention to the less preferred substrate. Growth in liquid cultures with dual provision of substrate such as this is often characterized by a second lag phase during the logarithmic growth period while this adaptation takes place. This is called diauxic growth (Fig. 3.11). As the association between bacteria and higher life forms becomes closer then more and more preformed biosynthetic building blocks become available without the need to synthesize them from their basic elements. Thus a pathogenic organism growing in soft tissues will have available to it glucose and metal ions from the blood and a whole plethora of amino acids, bases, vitamins, etc. from lysed tissue cells. While most bacteria will utilize these when they are available, a number of bacteria that have become specialized pathogens have lost their ability to synthesize many of these chemicals and so cannot grow in situations where the chemicals are not provided in the medium. Consequently many pathogens require complex growth media if they are to be cultured in vitro. 38
Growth on lactose Glucose exhausted Log growth
Time Fig. 3.11 Diauxic growth on a mixture of glucose and
lactose.
All that has been discussed earlier about the physicochemical and nutritional constraints on bacterial growth has been based on laboratory studies. By definition, the only bacteria that we can describe in this way are those that can be cultured artificially. It cannot be overstated that a majority of bacterial species and genera cannot be cultured in the laboratory. In the past the presence of such nonculturable bacteria has been attributed to dead, moribund cells. With the advent of modern molecular tools, however, it has now been realized that these organisms are viable. By amplifying their DNA and sequence mapping, the genetic relationship of such bacteria to the culturable ones can be demonstrated and whole new families of hitherto unrecognized bacteria are being identified. It is possible that in the future many disease states currently thought to have no microbiological involvement could be identified as being of bacterial origin. A recent example of this has been the association of Helicobacter pylori with gastric ulcers and gastric cancers.
8 Detection, identification and characterization of organisms of pharmaceutical and medical significance There are many situations in which microorganisms must not only be detected and enumerated but where they must also be identified either to make a
Bacteria
specific diagnosis of infection or to ensure the absence of specified bacteria from certain types of product. In such circumstances various cultural approaches are available that deploy enrichment and selection media. Once a microorganism has been isolated in pure culture, usually from a single colony grown on an agar plate, then further characterization may be made by the application of microscopy together with some relatively simple biochemical tests. Over the last 20 years the biochemical characterization of individual organisms has become simplified by the introduction of rapid identification systems. In recent years molecular approaches have enabled identification of organisms without the need to culture them. 8.1 Culture techniques Conventional approaches to microbiological examination of specimens require that they be cultured to assess the total numbers of specific groups of microorganisms or to determine the presence or absence of particular named species. The majority of samples taken for examination contain mixtures of different species, so simple plating onto an agar surface may fail to detect an organism that is present at < 2% of the total viable population. Various enrichment culture techniques may therefore be deployed to detect trace numbers of particular pathogens, prior to confirmatory identification. 8.1.1 Enumeration The simplest way in which to enumerate the microorganisms that contaminate an object or liquid sample is to dilute that sample to varying degrees and inoculate the surface of a pre-dried nutrient agar with known volumes of those dilutions (see Chapter 2). Individual viable bacteria that are able to grow on the nutrients provided and under the conditions of incubation will produce visible colonies that can be counted and the numbers related back to the original sample. Such counting procedures are often lengthy and tedious, the number of colonies formed might not relate to the viable number of cells, as clumps of cells will only produce a single colony and they will only detect a particular subset of the viable bacteria present in the sample
that can grow under the chosen conditions. Accordingly a variety of different media and cultural conditions are deployed to enumerate different categories of organism. A number of techniques are currently being developed in order speed up the enumeration process, although some of these rapid enumeration techniques indirectly measure the most probable number of viable cells. 8.1.1.1 Enumeration media Enumeration media will only ever culture a subset of cells towards which the medium and incubation conditions are directed. Thus, simple salts media with relatively simple sugars as carbon sources and trace levels of amino acids are often used to enumerate bacteria associated with water (e.g. R2A medium). Such plates may be incubated under aerobic or anaerobic conditions at a range of temperatures. Different temperatures will select for different subsets of cells, therefore any description of a viable bacterial count must specify the incubation conditions. In medical microbiology temperatures akin to the human body are often deployed because only those bacteria able to grow at such temperatures are likely to cause infection. However, psychrophilic Gram-negative bacteria (growing in water at 10°C) can be a major source of bacterial pyrogen, so a variety of incubation temperatures are often used in monitoring pharmaceutical waters and products. Highly nutritious media, e.g. blood agar, are also used as enumeration media. This is particularly the case when looking for microorganisms such as staphylococci that are usually found in association with animals and man. Such agar plates may be deliberately exposed to air (settle plates) and the number of colonies formed related to the bacteria content of a room. In the pharmaceutical industry microbiological monitoring will generally report the total aerobic count and, less commonly, the total anaerobic counts obtained on a moderately rich medium such as tryptone soya agar. Sometimes inhibitors of bacterial growth (e.g. Rose Bengal) can be added to a medium in order to select for moulds. 8.1.1.2 Rapid enumeration techniques The detection and quantification of components of 39
Chapter 3
bacterial cells is considerably faster than those approaches requiring the growth of colonies, and estimates of total viable cell number can thereby be obtained within minutes rather than hours and days. Some of the rapid methods that have been used for bacteria and other microorganisms, e.g. bioluminescence, epifluorescence and impedance techniques, have been described in Chapter 2, but there are other rapid methods that have found more limited application; these will be considered here. In the examination of pharmaceutical waters and aqueous pharmaceutical products electronic particle counters, e.g. Coulter counters, can be used to determine bacterial concentration, although these instruments do not discriminate between living and dead cells. Similar counters are available that are able to analyse particles found in air. Other rapid techniques aim to detect microbial growth rather than to visualize individual cells and colonies. As bacteria grow in liquid culture they not only alter the conductivity of the culture (see Chapter 2), they also generate small quantities of heat. The time taken to detect this heat can be directly related to the numbers of viable cells present by means of microcalorimeters. Once again this is a considerable improvement over conventional culture, but unlike particle counting and bioluminescence can only detect those organisms that are able to grow in the chosen medium. None of the rapid techniques are able to isolate individual organisms. They do not therefore aid in the characterization or identification of the contaminants. 8.1.2 Enrichment culture Enrichment cultures are intended to increase the dominance of a numerically minor component of a mixed culture such that it can be readily detected on an agar plate. Enrichment media are always liquid and are intended to provide conditions that are favourable for the growth of the desired organism and unfavourable for the growth of other likely isolates. This can be achieved either through manipulation of the pH and tonicity of the medium or by the inclusion of chemicals that inhibit the growth of unwanted species. Thus, MacConkey broth con40
tains bile salts that will inhibit the growth of nonenteric bacteria and may be used to enrich for Enterobacteriaceae. Several serial passages through enrichment broths may be made, and after enrichment it is not possible to relate the numbers of organisms detected back to that in the original sample. 8.1.3 Selective media Selective media are solidified enrichment broths, so again they are intended to suppress the growth of particular groups of bacteria and to allow the growth of others. The methods of creating this situation are the same as for enrichment broths. Thus mannitol salts agar will favour the growth of micrococci and staphylococci, and cetrimide agar will favour the growth of pseudomonads. The use of selective media is an adjunct to characterizing the nature of contaminants. Counts of colonies obtained on selective solid media are often documented as presumptive counts, so for example, colonies formed on a MacConkey agar (containing bile salts) might be cited as a presumptive coliform count. 8.1.4 Identification media (diagnostic) Identification media contain nutrients and reagents that indicate, usually through some form of colour formation, the presence of particular organisms. This enables them to be easily detected against a background of other species. In this fashion inclusion of lactose sugar and a pH indicator into MacConkey agar facilitates the identification of colonies of bacteria that can ferment lactose. Fermentation leads to a reduction in pH within these colonies and can be detected by an acid shift in the pH indicator, usually to red. Lactose-fermenting coliforms (Escherichia spp., Klebsiella spp.) can therefore be easily distinguished from non-fermentative coliforms (Salmonella spp., Shigella spp.). Similarly, the inclusion of egg-yolk lecithin into an agar gives it a cloudy appearance that clears around colonies of organisms that produce lecithinase (a virulence factor in staphylococci). While there are numerous types of selective and diagnostic media available, they can only be used as a guide to identi-
Bacteria
fication, but microscopy and biochemical or genetic characterization are much more definitive. 8.2 Microscopy Observation of stained and wet preparations of clinical specimens (blood, pus, sputum) and isolated pure cultures of bacteria from the manufacturing environment provides rapid and essential information to guide further identification. The application of simple stains such as the Gram stain can divide the various genera of bacteria into two convenient broad groups. The size and shapes of individual cells and their arrangement into clusters, chains and tetrads will also guide identification, as will specific stains for the presence of endospores, capsules, flagella and inclusion bodies. Examination of wet
preparations can give an indication as to the motility status of the isolate, and these procedures all represent an important first stage in the identification process. 8.3 Biochemical testing and rapid identification The differing ability of bacteria to ferment sugars, glycosides and polyhydric alcohols is widely used to differentiate the Enterobacteriaceae and in diagnostic bacteriology generally. Fermentation can be indicated by pH changes in the medium with or without gas production visualized by the collection of bubbles in inverted tubes. More specialized media examine the ability of certain strains to oxidize or reduce particular substrates. There are many
Table 3.3 Examples of some pharmaceutically useful bacteria Organism
Characteristics
Pharmaceutical relevance
Actinomyces spp. Bacillus stearothermophilus
Gram-positive, filamentous rods Gram-positive rod, aerobic, spore-former
Bacillus subtilis
Gram-positive rod, aerobic, spore-former
Bacillus pumilus
Gram-positive rod, aerobic, spore-former
Bordetella pertussis Brevundimonas (formerly Pseudomonas) diminuta Clostridium sporogenes
Gram-negative rod, aerobe Gram-negative, micro-aerobic rod
Antibiotic production Used to validate and monitor moist heat sterilization processes Used to validate and monitor dry heat and ethylene oxide sterilization processes Used to validate and monitor radiation sterilization processes Vaccine against whooping cough 0.22-µm filter challenge test
Clostridium tetani Corynebacterium diphtheriae Escherichia coli Haemophilus influenzae type b Leuconostoc mesenteroides Neisseria meningitidis Pseudomonas aeruginosa Proteus vulgaris Salmonella typhi Staphylococcus aureus
Gram-positive rod, anaerobe, spore-former Gram-positive rod, anaerobe, spore-former Gram-positive rod, aerobe Gram-negative enteric rod, facultative anaerobe Gram-negative rod, aerobe Gram-positive rod Gram-negative cocci, aerobic Gram-negative, micro-aerobic rod Gram-negative, aerobic rod Gram-negative enteric rod, facultative anaerobe Gram-positive, aerobic cocci
Used to confirm anaerobic growth conditions Vaccine against tetanus Vaccine against diphtheria Kelsey-Sykes disinfectant capacity test Preservative limit test Vaccine against Hib infections Dextran production Vaccine against meningitis C Alginate production Kelsey-Sykes disinfectant capacity test Kelsey-Sykes disinfectant capacity test Chick Martin/Rideal Walker disinfectant coefficient test Kelsey-Sykes disinfectant capacity test Preservative limit test
41
Chapter 3 Table 3.4 Examples of some pharmaceutically problematic bacteria Organism
Characteristics
Pharmaceutical relevance
Bacteroides fragilis Bordetella pertussis Campylobacter jejuni
Gram-negative enteric rod, anaerobe Gram-negative rod, aerobe Gram-negative enteric spiral rod, micro-aerophilic Clostridium tetani Gram-positive rod, anaerobe, spore-former Corynebacterium diphtheriae Gram-positive rod, aerobe Escherichia coli Gram-negative enteric rod, facultative anaerobe Haemophilus influenzae Gram-negative rod, aerobe Legionella pneumophila Mycobacterium tuberculosis
Gram-negative rod, aerobic Gram-positive, acid-fast rod, aerobe
Pseudomonas aeruginosa
Gram-negative, micro-aerobic rod
Salmonella spp. Staphylococcus aureus
Gram-negative enteric rods, facultative anaerobes Gram-positive, aerobic, catalase-positive cocci
Staphylococcus epidermidis
Gram-positive, aerobic, catalase-positive cocci
Streptococcus spp.
Gram-positive, aerobic, catalase-negative cocci
hundreds of individual biochemical tests available that each separately seek the presence of a particular enzyme or physiological activity. Taxonomic studies have led to the recognition that certain of these tests in combination characterize particular species of bacteria. Various manuals such as Bergey’s Manual and Cowan and Steele’s Manual for the Identification of Medically Important Bacteria provide a logical and sequential framework for the conduct of such tests. Identification of particular species and genera by such processes is timeconsuming, expensive and may require numerous media and reagents. This process has become simplified in recent years by the development of rapid identification 42
Wound infections Causative agent of whooping cough Severe enteritis Causative agent of tetanus Causative agent of diphtheria Food poisoning, severe enteritis Causative agent of infantile meningitis and chronic bronchitis Causative agent of Legionnaire’s disease Causative agent of tuberculosis Disinfectant resistance Intracellular pathogen General environmental contaminant Quintessential opportunist pathogen High resistance to antibiotics and biocides Biofilm-former Varying degrees of food poisoning, typhoid fever Skin contaminant Food poisoning Toxic shock syndrome Pyogenic infections Implanted medical device/prosthetic device contaminant Biofilm-former Causative agents of tonsilitis and scarlet fever
methods and kits. The latter often use multi-well microtitration plates that can be inoculated in a single operation either with an inoculated wire or with a suspension of a pure culture. Each individual well contains the medium and reagents for the conduct of a single biochemical test. Identification kits vary in their complexity and also in the precision of the identification made. Simple kits may perform only 8–15 tests, more complex ones are capable of performing 96 simultaneous biochemical evaluations. Scoring of each test and entry into a computer database then allows the pattern of test results to be compared with a large panel of organisms and a probability of identity calculated. As different sets of tests will be required for different classes of bac-
Bacteria
teria, guidance as to the initial choice of kit is given on the basis of the Gram stain reaction, and the results of oxidase and catalase tests performed directly on isolated colonies. In large diagnostic laboratories and in quality assurance laboratories automated systems are deployed that can inoculate, incubate and analyse hundreds of individual samples at a time. 8.4 Molecular approaches to identification The need to identify microorganisms rapidly has led to the development of a number of molecular identification and characterization tools. These have not yet become routinely adopted in the analytical or diagnostic laboratory but will probably do so in the future. One such technique isolates and amplifies 16S ribosomal DNA and, following sequencing of the bases, compares this with known sequences held in a reference library. This approach enables phylogenetic relationships to be derived even for those bacteria that have not previously been identified. Other systems examine the patterns of key constituents of the cells such as fatty acids and assign identities based on similarity matches to known reference cultures. Molecular approaches can be of particular use when attempting to detect a particular species. Thus gene probes carrying fluorescent dyes can be used in hybridization procedures with the collected clinical material. Examination under the fluorescent microscope will show the targeted organism as fluorescent against a background of nonfluorescent organisms. 8.5 Pharmaceutically and medically relevant microorganisms Microorganisms of medical and pharmaceutical relevance can be broadly classified into those organisms that are harmful or problematic, and those that can be used to our advantage. Some microor-
ganisms, depending on the situation, can fall into both categories. Microorganisms cause some of the most important diseases of humans and animals and they can also be found as major contaminants of pharmaceutical products. On the other hand, many large-scale industrial processes, e.g. antibiotic production, are based on microorganisms, and selected species can be used to test disinfectant efficacy and to monitor sterilization procedures. Tables 3.3 and 3.4 respectively, list examples of some of the more pharmaceutically relevant beneficial and problematic microorganisms. Specific texts should be referred to for more detailed descriptions.
9 Further reading Buchanan, R. E. & Gibbons, N. E. (1974) Bergey’s Manual of Determinative Bacteriology, 8th edn. Williams & Wilkins, Baltimore. Costerton, J. W., Lewandowski, Z., deBeer, D., Caldwell, D., Korber, D. & James, G. (1994) Biofilms, the customised microniche. Journal of Bacteriology, 176, 2137–2142. Cowan, S. T. (1993) Cowan and Steel’s Manual for the Identification of Medical Bacteria (eds G. Barrow & R. K. A. Feltham), 3rd edn. Cambridge University Press, Cambridge. Gould, G. W. (1985) Modification of resistance and dormancy. In: Fundamental and Applied Aspects of Bacterial Spores (eds G. J. Dring, D. J. Ellar & G. W. Gould), pp. 371–382. Academic Press, London. Madigan, M. T., Martinko, J. M. & Parker, J. (2000) Brock Biology of Microorganisms, 9th edn. Prentice-Hall, New Jersey. Murray, P .R., Kobayashi, G. S., Pfaller, M. A. & Rosenthal, K. S. (1994) Medical Microbiology, 2nd edn. Mosby-Year Book, London. Roitt, I. M. (1994) Essential Immunology, 8th edn. Blackwell Scientific Publications, Oxford. Salyers, A. A. & Drew, D. D. (1994) Bacterial Pathogenesis: A Molecular Approach. ASM Press, Washington. Stryer, L. (2002) Biochemistry, 5th edn. W. H. Freeman & Co., San Francisco. Sutherland, I. W. (1985) Biosynthesis and composition of Gram-negative bacterial extracellular and wall polysaccharides. Annu Rev Microbiol, 10, 243–270.
43
Chapter 4
Fungi Kevin Kavanagh and Derek Sullivan 1 2 3 4 5
What are fungi? The structure of the fungal cell Medical significance of fungi Fungal species identification methods Antifungal therapy 5.1 Polyene antifungal agents 5.2 Azole antifungal agents 5.3 Synthetic antifungal agents 6 Mechanisms of antifungal drug resistance
1 What are fungi? Yeasts, such as brewers’ yeast, and moulds, such Penicillium chrysogenum which produces the antibiotic penicillin, are classified as fungi. Yeast cells tend to grow as single cells that reproduce asexually in a process known as budding, although a minority of species (e.g. Schizosaccharomyces pombe) reproduce by fission. Many yeast species are capable of sexual reproduction and the formation of spores. In contrast, moulds grow as masses of overlapping and interlinking hyphal filaments and reproduce by producing masses of spores in a variety of structures. This division between yeasts and moulds based on growth morphology is not clear-cut, as some yeasts can produce hyphae under specific conditions (e.g. Candida albicans), while many normally filamentous fungi possess a yeastlike phase at some point in their life cycle. Fungi are eukaryotic organisms, i.e. their cells possess a nuclear membrane, consequently there are many similarities between the biochemistry of fungal cells and human cells. Fungi are widely distributed in nature, occurring as part of the normal flora on the body of warm-blooded animals, as decomposers of organic matter and as animal and plant pathogens. Medically, fungi are an extremely important group of microbes, being responsible for a number of potentially fatal diseases in humans (Table 4.1), but a
7 Some clinically important fungi 7.1 Candida albicans — the dominant fungal pathogen 7.2 Aspergillus fumigatus 7.3 Histoplasma capsulatum 7.4 Cryptococcus neoformans 7.5 Dermatophytes 8 Antibiotic production by fungi 9 Genetic manipulation of fungi 10 Further reading
significant number of fungi are of great benefit to humanity in terms of the production of alcoholic beverages, bread and antibiotics (Table 4.2). Fungi have also been utilized for a range of molecular biological applications. From a taxonomic point of view the Kingdom fungi can be subdivided into six Classes. The Class Oomycetes contains the mildews and water moulds, the Class Ascomycetes contains the mildews, some moulds and most yeast species (including Saccharomyces cerevisiae), the Class Basidiomycetes contains the mushrooms and bracket fungi, the Class Teliomycetes contains the rust fungi (plant pathogens), the Class Ustomycetes contains the smuts (plant pathogens) and the Class Deuteromycetes contains species such as Aspergillus, Fusarium and Penicillium (Fig. 4.1 illustrates the typical septate hyphae of this Class).
2 The structure of the fungal cell The typical yeast cell is oval in shape and is surrounded by a rigid cell wall, which contains a number of structural polysaccharides and may account for up to 25% of the dry weight of the cell wall (Fig. 4.2). Glucan accounts for 50–60%, mannan for 15–23% and chitin for 1–9% of the dry weight of the wall, respectively, with protein and lipids also 44
Fungi Table 4.1 Examples of fungal diseases and selected causative agents Type of mycosis
Disease
Species name
Superficial
Pityriasis versicolor White piedra Tinea pedis (athlete’s foot) Onychomycosis (nail infection) Tinea capitis (scalp ringworm) Chromoblastomycosis Mycetoma Blastomycosis Histoplasmosis Coccidioidomycosis Paracoccidioidomycosis Candidosis (superficial/systemic)
Malassezia furfur Trichosporon beigelii Trichophyton rubrum Trichophyton rubrum Trichophyton tonsurans Fonsecaea pedrosoi Acremonium spp. Blastomyces dermatitidis Histoplasma capsulatum Coccidioides immitis Paracoccidioides brasiliensis Candida albicans Candida glabrata Candida parapsilosis Aspergillus fumigatus Pneumocystis carinii
Cutaneous
Subcutaneous Systemic
Opportunistic
Aspergillosis Pneumonia
Table 4.2 Examples of economically important fungi Fungal species Filamentous fungi Agaricus bisporus Aspergillus, Penicillium spp. Aspergillus sp. + Saccharomyces sp. Fusarium graminearum Penicillium chrysogenum Penicillium notatum Penicillium roqueforti Yeasts Pichia sp. Saccharomyces cerevisiae
Application Edible mushroom Enzymes (catalase, lipase, amylase) Sake (rice wine) Single cell protein Penicillin production Enzyme (glucose oxidase) Cheese flavouring (Roquefort ‘blue’ cheese) Gene expression system Bakers’ yeast—bread Brewers’ yeast—beer, wine, cider, etc. Enzyme (invertase) Gene expression system Dietary supplement
present in smaller amounts. The thickness of the cell wall may vary during the life of the cell but the average thickness in the yeast C. albicans varies from 100 to 300 nm. Glucan, the main structural compo-
Fig. 4.1 Septate hyphae of Aspergillus fumigatus.
nent of the fungal cell wall, is a branched polymer of glucose which exists in three forms in the cell, i.e. b-1,6-glucan, b-1,3-glucan and b-1,3,-b-1,6complexed with chitin. Mannan is a polymer of the sugar mannose and is found in the outer layers of the cell wall. The third principal structural component, chitin, is concentrated in bud scars which are areas of the cell from which a bud has detached. Proteins and lipids are also present in the cell wall and under some conditions may represent up to 30% of the cell wall contents. Mannoproteins form a fibrillar layer that radiates from an internal skeletal layer, which is formed by the polysaccharide component of the cell wall. The innermost layer is rich in glucan and chitin, which provides rigidity to the wall and is important in regulating cell division. 45
Chapter 4 Vacuole
Vacuolar granules
1µm
Mitochondrion
Nucleus
Cell wall Vacuolar membrane Storage granules
Bud vacuole
Pore in nuclear membrane Bud
Bud scar
Cell membrane
Enzymatic or mechanical removal of the cell wall leaves an osmotically fragile protoplast that will burst if not maintained in an osmotically stabilized environment. Incubation of protoplasts in an osmotically stabilized agar growth medium will allow the re-synthesis of the wall and the resumption of normal cellular functions. The ability to generate protoplasts from fungi opens the possibility of fusing these under defined conditions to generate strains with novel biotechnological applications. The periplasmic space is a thin region that lies directly below the cell wall. It contains secreted proteins that do not penetrate the cell wall and is the location for a number of enzymes required for processing nutrients before entry into the cell. The cell membrane or plasmalemma is located directly below the periplasmic space and is a phospholipid bilayer that contains phospholipids, lipids, protein and sterols. The plasmalemma is approximately 10 nm thick and in addition to being composed of phospholipids also contains globular proteins. The dominant sterol in fungal cell membranes is ergosterol, which is the target of the antifungal agent amphotericin B. Sterols are important components of the plasmalemma and represent regions of rigidity in the fluidity provided by the phospholipid bilayer. Most of the cell’s genome is concentrated in the nucleus, which is surrounded by a nuclear membrane that contains pores to allow communication with the rest of the cell (Fig. 4.2). The nucleus is a discrete organelle and, in addition to being the repository of the DNA, also contains proteins in the 46
Fig. 4.2 Diagrammatic
representation of ‘typical’ yeast cell.
form of histones. Yeast chromosomes vary in size from 0.2 to 6 Mb and the number per yeast is also variable, with S. cerevisiae having as many as 16 while the fission yeast Sch. pombe has as few as 3. In addition to the genetic material in the nucleus the yeast cell often has extrachromosomal information in the form of plasmids. For example, the 2-mm plasmid is present in S. cerevisiae, although its function is unclear, and killer plasmids in the yeast Kluyveromyces lactis which encode a toxin. Actively respiring fungal cells possess a distinct mitochondrion, which has been described as the ‘power-house’ of the cell (Fig. 4.2). The enzymes of the tricarboxylic acid cycle (Kreb’s cycle) are located in the matrix of the mitochondrion, while electron transport and oxidative phosphorylation occur in the mitochondrial inner membrane. The outer membrane contains enzymes involved in lipid biosynthesis. The mitochondrion is a semiindependent organelle as it possesses its own DNA and is capable of producing its own proteins on its own ribosomes, which are referred to as mitoribosomes. The fungal cell contains a vast number of ribosomes, which are usually present in the form of polysomes — lines of ribosomes strung together by a strand of mRNA. Ribosomes are the site of protein biosynthesis. The system that mediates the export of proteins from the cell involves a number of membranous compartments including the Golgi apparatus, the endoplasmic reticulum and the plasmalemma. In addition, the vacuole is employed as a ‘storage space’ where nutrients, hydrolytic enzymes
Fungi
or metabolic intermediates are retained until required.
3 Medical significance of fungi Fungi represent a significant group of pathogens capable of causing a range of diseases in humans. While the majority of fungi appear to be harmless to humans it is worth bearing in mind that in the right set of conditions a normally non-pathogenic fungus can cause a clinically relevant problem. In a case of a profoundly immunocompromised individual, a wide range of fungi can present as capable of inducing disease. The most common fungal pathogens of humans can be divided into three broad classes: the yeasts, the moulds and the dermatophytes. The yeast C. albicans is the most frequently encountered human fungal pathogen, being responsible for a wide range of superficial and systemic infections. The superficial infections include oropharyngeal and genital conditions, the former occur predominantly in HIV-positive individuals, geriatric people and premature infants and may arise when a weakened or immature immune system is present. Genital candidosis is very common and approximately 75% of women are affected by vulvovaginal candidosis (VVC) during their life, with a further 5–12% suffering from recurring bouts of infection over a prolonged period of time. Genital candidosis in men is comparatively rare, although it is occasionally seen in alcoholics and diabetics. Candidosis of the skin and fingernails (onychomycosis) may be present in up to 10% of cancer patients. Continually moist, macerated or burnt skin is an ideal environment for the development of cutaneous candidosis. C. albicans is also responsible for a range of systemic infections and many of these can commence as superficial infections. Infection of the gastrointestinal tract is seen in diabetics, cancer and AIDS patients and the oesophagus is a common site of infection, rendering swallowing difficult. Candida infection of the respiratory tract is seen in those with underlying disease (e.g. cancer) or following the insertion of a silicone voice prosthesis. The urinary tract can also be the site of candidosis, which may be due to renal infection, other underlying dis-
ease(s) or cystitis. The presence of an indwelling urinary catheter may also predispose to Candida infection. A range of factors is capable of predisposing the individual to superficial or systemic infections. Factors which impair the host’s immune system such as the presence of underlying disease (AIDS, cancer, diabetes), the use of immunosuppressive therapy during organ transplantation and broad-spectrum antibiotic therapy can leave the individual susceptible to candidosis. Different stages of life may also predispose to specific types of infection. For example, pregnant women have a higher incidence of vaginal candidosis and premature infants and geriatric individuals are susceptible to oral candidosis due to either immature or faltering immune systems, respectively. Other factors that may predispose to Candida infection include the presence of indwelling catheters and skin damage as a result of burns or other trauma. The mould Aspergillus fumigatus is the dominant fungal pulmonary pathogen of humans and generally presents as a problem in those with preexisting lung disease or damage. In addition to pulmonary infection other sites may be affected including the brain, kidneys and sinuses, depending on the level of immunocompromisation of the individual. Groups particularly susceptible to colonization by Aspergillus species include those with cavities as a result of tuberculosis, patients affected with asthma or cystic fibrosis and those with profound immunosuppression due to leukaemia (neutropenia). Aspergillosis presents as a serious problem in patients immunosuppressed in advance of organ transplantation. Dermatophyte is the term applied to a range of fungi capable of colonizing the skin, nails or hair. The principal dermatophytic fungi are Trichophyton, Microsporum and Epidermophyton species. The most commonly encountered dermatophytic infections are athlete’s foot (infection of the foot) and ringworm (fungal infection of the scalp or skin).
4 Fungal species identification methods The first step in identifying a fungus in clinical sam47
Chapter 4
ples is to observe the tissue sample microscopically, often in conjunction with specific staining procedures. For instance, addition of potassium hydroxide to the specimen facilitates any fungal elements present to be distinguished from host structures. Tissue samples routinely tested for fungi in the diagnostic laboratory include blood, cerebrospinal fluid, bronchial aspirates and biopsy material, all of which are usually sterile. While microscopic analysis of samples is certainly rapid and inexpensive it is not very sensitive. Consequently, in the diagnostic laboratory samples are routinely inoculated onto agar plates that specifically allow the growth of fungi. A wide variety of media have been developed for the selective growth of fungal species. On these media filamentous fungi are often identifiable to the species level based on their colony morphology following macroscopic and microscopic analysis, with many species having recognizable characteristic hyphal and conidial structures. As C. albicans and the recently discovered closely related species C. dubliniensis can grow in the yeast or hyphal form, the ability of these species to produce germ-tubes (i.e. hyphae) can easily be tested in the laboratory by incubation of the cells in serum. Similarly these species are also the only members of the genus Candida to produce large spore-like structures called chlamydospores on specific media. Identification of other clinically important Candida species has been greatly facilitated by the recent introduction of commercially available chromogenic media, which allow the Candida species to be distinguished from each other on the basis of colony colour. In addition, Candida species can also be identified on the basis of their different nutrient (especially carbohydrate) requirements, which can be assessed using commercial kits. In addition, as in the case of direct microscopic analysis of samples, the levels of fungal elements present in samples taken are often very low and are not present at sufficiently high levels to be detected using culture-based methods. Therefore, in many cases the identity of the agent responsible for causing a fungal infection is only determined once the infection has been cured using empirical treatment. Indeed sometimes the cause of disease is only identified at autopsy. Clearly, to improve our ability to treat fungal diseases effectively there is a great need to develop 48
techniques that allow the detection and identification of infecting fungal species more quickly. Currently, two areas of research are endeavouring to facilitate this goal. Firstly, it has been proposed that rather than detecting the infecting species it may be more appropriate to monitor the host immune response to specific pathogens. To this end enzymelinked immunosorbent assay (ELISA)-based tests have been developed to detect antibodies to specific antigens belonging to fungal species. In another recent development molecular biologists are attempting to adapt polymerase chain reaction (PCR)-based methods to detect the nucleic acids of specific fungal species in normally sterile sites. This method in tandem with genome-based microchip technology offers great promise for automated diagnosis. While both ELISA-based and PCR-based technologies have the potential to revolutionize the diagnosis of fungal infections as they are very rapid and accurate, they are still at the experimental stage and are only in use in specialized laboratories.
5 Antifungal therapy The choice and dose of an antifungal agent will depend on the nature of the condition, whether there are any underlying diseases, the health of the patient and whether antifungal resistance has been identified as compromising therapy. Part of the difficulty in designing effective antifungal agents lies in the fact that fungi are eukaryotic organisms, so agents that will kill fungi may also have a deleterious effect on human tissue. The ideal antifungal drug should target a pathway or process specific to the fungal cell, so reducing the possibility of damaging tissue and inducing unwanted side-effects. 5.1 Polyene antifungal agents Polyene antifungal agents are characterized by having a large macrolide ring of carbon atoms closed by the formation of an internal ester or lactone (Fig. 4.3). In addition, polyenes have a large number of hydroxyl groups distributed along the macrolide ring on alternate carbon atoms. This combination of highly polar and non-polar regions within the molecule render the polyenes amphiphatic, i.e.
Fungi CH3
O H
H3C
OH NH2
O OH H
CH3
HO
O
OH
OH
OH
OH
O
H3C
COOH
O
OH
OH
OH Amphotericin B N N
Cl
N Cl
O
O
CH3
O
CH3
N N
O
H
N
N
N
Itraconazole Cl OH
N N
O
N
N
N
N
N
F
N Cl
Cl
F Fluconazole
Miconazole
Cl
O
O Cl O Fig. 4.3 Structures of polyene
(amphotericin B) and azole (itraconazole, fluconazole, miconazole and ketoconazole) antifungal agents.
having hydrophobic and hydrophilic regions in the one molecule which assists solubility in lipid membranes. The principal polyenes are amphotericin B and nystatin. Amphotericin B is produced by the bacterium Streptomyces nodosus and its activity is due to the ability to bind ergosterol, which is the dominant sterol in fungal cell membranes, and consequently increases membrane permeability by the formation of pores (Fig. 4.4). The action of ampho-
Cl
O
N
N
CH3
H
N N
Ketoconazole
tericin B seems to rely on the formation of pores through which intracellular contents can escape from the cell. Amphotericin B can lead to renal damage during prolonged antifungal therapy. Amphotericin B is active against a broad range of fungal pathogens and is considered the ‘gold standard’ against which the activity of other antifungal agents is measured. Due to its renal toxicity amphotericin B tends to be reserved for severe cases of systemic fungal disease, but recent formulations in which the 49
Chapter 4 CH3
H3C CH3
Lanosterol
CH3
CH3 CH3 HO H3C
CH3 14-a-demethylase
Site of action of azoles
14-a-demethyl-Lanosterol
CH3
CH3
HO H3C
CH3
H 3C CH3
CH3
Accumulation of ergosterol precursors CH3 CH3
H 3C CH3
Ergosterol CH3
CH3
H HO Site of action of amphotericin B
drug is encapsulated within liposomes have been shown to be of reduced toxicity. Nystatin was discovered in 1950 and exhibits the same mode of action as amphotericin B but tends to be of lower solubility, which has restricted its use to the treatment of topical infections. While nystatin was effective for the treatment of conditions such as oral and vaginal candidosis its use has been overtaken by the introduction of azole antifungal drugs. 5.2 Azole antifungal agents The first generation of azole antifungal agents revo50
Fig. 4.4 Modes of action of polyene and azole antifungal agents showing sites of action.
lutionized the treatment of mucosal and invasive fungal infections and azoles are still the most widely used group of antifungal agents. The azole derivatives are classified as imidazoles or triazoles on the basis of whether they have two or three nitrogen atoms in the five-membered azole ring (Fig. 4.3). The azoles in current and long-standing clinical use are clotrimazole, miconazole, econazole and ketoconazole, while newer drugs such as itraconazole, fluconazole and voriconazole have important applications in the treatment of systemic infections. Azoles function by interfering with ergosterol biosynthesis by binding to the cytochrome P-450-
Fungi
mediated enzyme known as 14-a-demethylase (P450DM). This blocks the formation of ergosterol by preventing the methylation of lanosterol (a precursor of ergosterol) (Fig. 4.4), resulting in a reduction in the amount of ergosterol in the fungal cell membrane, which leads to membrane instability, growth inhibition and cell death in some cases. An additional consequence of the block in ergosterol biosynthesis is the build-up of toxic intermediates, which can prove fatal to the cell (see Chapter 12). Azoles exhibit a broad spectrum of activity in vitro, being capable of inhibiting the growth of most Candida, Cryptococcus and Aspergillus species and dermatophytes. Miconazole was the first azole used to treat systemic fungal infections but demonstrated a number of toxic side-effects. Ketoconazole produced high serum concentrations upon oral administration but had poor activity against aspergillosis. In addition, ketoconazole was associated with a range of side-effects which limited its applicability. Newer triazoles such as fluconazole and itraconazole have increased the options for dealing with fungal infections. Fluconazole was introduced for clinical use in 1990, is water-soluble and shows good penetration and deposition into the pulmonary tissues; it also reaches high levels in the cerebrospinal and peritoneal fluids. Fluconazole has proved highly effective in the treatment of infections caused by C. albicans but shows limited activity against Aspergillus. Itraconazole became available for clinical use in the late 1980s and was the first azole with proven efficacy against Aspergillus. Itraconazole is effective in treating severe Aspergillus infections and exhibits both fungicidal and fungistatic effects. Upon ingestion itraconazole undergoes extensive hepatic metabolism which yields up to 30 metabolites — a number of which retain antifungal activity. Itraconazole is currently available as an intravenous formulation and is widely used for the treatment of severe Aspergillus infection in this form. Fluconazole and itraconazole demonstrate significantly reduced side-effects compared with ketoconazole. Novel azole drugs with increased ability to inhibit the fungal 14-ademethylase are also becoming available. These agents, which include voriconazole, posaconazole and ravuconazole, have a wider spectrum of activity than fluconazole and it has been suggested that
some of them show fungicidal effects with some species (e.g. Aspergillus spp.). Voriconazole is one of the newest second generation triazole antifungal drugs and it shows good activity against pulmonary aspergillosis and cerebral aspergillosis. 5.3 Synthetic antifungal agents Flucytosine is a synthetic fluorinated pyrimidine that has been used as an oral antifungal agent and demonstrates good activity against a range of yeast species and moderate levels of activity against Aspergillus species. Two modes of action have been proposed for flucytosine (see Chapter 12). One involves the disruption of protein synthesis by the inhibition of DNA synthesis, while the other possible mode of action is the depletion in the amino acid pools within the cell as a result of inhibition of protein synthesis. In general yeast cells increase in size when exposed to sub-MIC levels of flucytosine and display alterations in their surface morphology, both of which can be interpreted as a result of an imbalance in the control of cellular growth. Many fungi are inherently resistant to flucytosine or develop resistance after a relatively short exposure and resistance has been attributed to alteration in the enzyme (cytosine deaminase) required to process flucytosine once inside the cell or to an elevation in the amount of pyrimidine synthesis. The problem of resistance has limited the use of flucytosine so that now it is generally used in combination with an antifungal agent (e.g. amphotericin B) where it can potentiate the effect of the second agent.
6 Mechanisms of antifungal drug resistance Resistance to antifungal drugs is becoming an increasingly perplexing problem. This is particularly the case with one of the most widely used antifungal agents, fluconazole. As outlined previously this drug is a fungistatic agent that acts by inhibiting the activity of cytochrome P-450 (lanosterol ademethylase), an enzyme in the pathway responsible for the synthesis of ergosterol. Inhibition of this enzyme results in decreased ergosterol levels and a build-up of toxic intermediary products, 51
Chapter 4
which subsequently leads to aberrant membrane function and stasis of cell growth and division. However, since the widespread introduction of fluconazole during the early 1990s, there have been increasing reports of Candida strains that have decreased susceptibility to azole drugs and of infections that are recalcitrant to treatment. In addition, the prevalence of infections caused by Candida species that are inherently less susceptible to azoles has increased over the same time period. One of the most important mechanisms of resistance is the overexpression of efflux proteins which act by pumping the drug out of the cell at a rate faster than the drug enters the cell (see Fig. 4.5). Therefore the intracellular concentration of the drug does not reach a level high enough to inhibit the target enzyme. Another resistance mechanism that has been identified is mutation within the gene encoding the cytochrome P-450. These amino acid substitutions do not affect the activity of the enzyme, but the conformation of the enzyme is sufficiently altered to decrease the affinity of the enzyme for the drug, thus the levels of membrane ergosterol are not altered significantly. An alternative resistance mechanism is the overexpression of the gene encoding the target cytochrome enzyme, this results in increased levels of the cytochrome inside the cell, thus countering the effects of the drug. Finally, another resistance mechanism is mutation in genes that encode other proteins involved in the biosynthesis of ergosterol. These mutations prevent the build-up of toxic byproducts, thus the inhibitory effects of the azoles are decreased. Very often in azole-resistant clinical isolates of C. albicans multiple resistance mechanisms have been identified as contributing to the resistance phenotype, thus it is difficult to determine which are the most important mechanisms (see Fig. 4.5). Resistance to other antifungal agents such as amphotericin B has been observed less frequently in clinical fungal isolates; however, the molecular basis of this resistance is not currently well understood. To solve the problems associated with antifungal resistance a number of novel azole drugs (e.g. voriconazole) and novel classes of drug (e.g echinocandins) have been developed (see Chapter 12).
52
7 Some clinically important fungi 7.1 Candida albicans — the dominant fungal pathogen The yeast C. albicans is an opportunist fungal pathogen which can be present as a normal part of the body’s microflora. C. albicans displays a variety of virulence factors which aid colonization and persistence in the body. One of the most important of these factors is the ability to adhere to host tissue using a variety of mechanisms (Fig. 4.6). The importance of adherence may be illustrated by the ability of C. albicans to adhere to various mucosal surfaces and to withstand forces that may lead to its removal from the body such as the bathing/washing action of body fluids. A hierarchy exists among Candida species indicating that the more common aetiological agents of candidosis (C. albicans and Candida tropicalis) are more adherent to host tissue in vitro than relatively non-pathogenic species such as C. krusei and Candida guilliermondii. Adherence to host tissue is achieved by a combination of specific and non-specific mechanisms. Specific adherence mechanisms include ligand– receptor interactions, while non-specific mechanisms include electrostatic forces, aggregation and cell surface hydrophobicity. Non-specific interactions are the primary interaction (occurring over longer distances) involved in the adherence process and are reversible. However, non-specific adherence is consequently deemed irreversible as a result of specific mechanisms that involve the ability of the yeast to recognize a variety of host cell receptors/ ligands using cell surface molecules. C. albicans can exist in two morphologically distinct forms — budding blastospores or hyphae. The yeast can switch between each form and is usually encountered in tissue samples in both morphological forms (Fig. 4.7). The hyphae are capable of thigmotropism (contact sensing), which may aid in finding the line of least resistance between and through layers of cells in tissue. C. albicans produces a range of extracellular enzymes that facilitate adherence and/or tissue penetration. Phospholipase A, B, C and lysophospholipase may function to damage host cell membranes and facilitate invasion. C. albicans produces a range of acid
Fungi
Fluconazole
Lanosterol
Lanosterol
Cytochrome
Cytochrome
Ergosterol
Toxic by-products
(a)
(b)
Lanosterol
Lanosterol
(c)
(d)
Cytochrome
Ergosterol
Ergosterol
(e)
Fig. 4.5 Mechanisms of resistance to azole drugs commonly found in Candida species. (a) A major step in ergosterol synthesis is
catalysed by the cytochrome P-450 enzyme lanosterol 14a-demethylase. (b) Fluconazole inhibits the activity of cytochrome P-450 enzyme lanosterol 14a-demethylase, which results in a depletion of the ergosterol content of the cell membrane and the build-up of toxic precursors. (c) Overexpression of the gene encoding the lanosterol 14a-demethylase allows enough enzyme activity to enable sufficient ergosterol production. (d) Mutations in the active site of the target enzyme can result in a decreased affinity for the drug without affecting the catalytic activity of the enzyme. (e) Increased expression of the genes encoding specific transmembrane pumps (e.g. CDR1 and MDR1) prevents the accumulation of the drug inside the cell.
53
Chapter 4
proteinases which have been shown to aid adherence and invasion but which also play an important role in the degradation of the immunoglobulins IgG and IgA. The proteinases have a low pH optimum and this may assist the yeast colonization of the vagina, which is a low pH environment. Haemolysin production by C. albicans has also been documented and seems to be important in allowing the yeast access to iron released from ruptured red blood cells. An important immune evasion tactic of C. albicans is the ability to bind to platelets via fibrinogen-binding ligands, which results in the fungal cell being surrounded by a cluster of platelets. C. albicans is capable of giving rise to a variety of inter-convertible phenotypes which can be consid-
Fig. 4.6 Cells of C. albicans (arrows) adhering to a human buccal epithelial cell. Y, yeast cells; H, hyphal cells.
ered as providing an extra dimension to the existing virulence factors associated with this yeast. A number of switching systems have been identified and phenotypic switching may have evolved to compensate for the lack of variation achieved in other organisms that utilize sexual reproduction. Phenotypic switching allows the yeast to exploit micro-niches in the body and alters a variety of factors (e.g. antifungal drug resistance, adherence, extracellular enzyme production), in addition to the actual phenotype and so may be considered as the ‘dominant’ or ‘controlling’ virulence factor. In terms of tissue colonization and invasion, adherence is the initial step in the process. Once the yeast has adhered, enzymes (phospholipase and proteinase) can facilitate adherence by damaging or degrading cell membranes and extracellular proteins. Hyphae may be produced and penetrate layers of cells using thigmotropism to find the line of least resistance. The passage through cells is undoubtedly aided by the production of extracellular enzymes. Once endothelial cells are reached enzymes may assist in the degradation of tissue and allow the yeast to enter the host’s bloodstream, where phenotypic switching or coating with platelets may be used to evade the immune system. While in the bloodstream the haemolysin may function to burst blood cells and release iron, which is essential for growth. Escape from the bloodstream involves adherence to the walls of capillaries and passage across the wall.
A B D C Fig. 4.7 Growth morphologies of
Candida albicans. (A) Budding morphology; (B) hyphal formation; (C) germ-tube formation leading to hyphal formation; (D) pseudohyphal formation.
54
Fungi
7.2 Aspergillus fumigatus Aspergillus fumigatus is a saprophytic fungus that is widely distributed in nature where it is frequently encountered growing on decaying vegetation and damp surfaces. A. fumigatus can present as an opportunist pathogen of humans and is the commonest aetiological agent of pulmonary aspergillosis, being responsible for 80–90% of cases. While the incidence of disease due to Aspergillus species is less than that due to Candida, aspergillosis results in greater mortality rates. A. fumigatus produces a number of extracellular enzymes that facilitate growth in the lung and dissemination through the body. Phospholipase production has been shown in clinical isolates of this fungus, with optimal production occurring at 37°C. This enzyme plays a critical role in tissue degradation and may facilitate exit of the fungus from the lung into the bloodstream. Fungal proteases are responsible for tissue degradation and neutralization of the immune system and probably evolved to allow the fungus to degrade animal and plant material. Elastin constitutes almost 30% of lung tissue and many A. fumigatus isolates display elastinolytic activity, while isolates incapable of elastinase production display reduced virulence in mice. A. fumigatus produces two elastinases: a serine protease and a metalloproteinase. Apart from their direct role in tissue degradation A. fumigatus proteases (in particular the serine protease) may also function as allergens, which may be important in the induction and persistence of allergic aspergillosis. Local inflammation due to the presence of proteases results in airway damage and in vitro studies have shown that proteases are capable of inducing epithelial cell detachment from basement membranes. Such desquamation may partly explain the extent of damage seen in the lungs of asthmatic and cystic fibrosis patients affected with allergic aspergillosis. In addition, proteases induce the release of the pro-inflammatory IL-6 and IL-8 cytokines in cell lines derived from airway epithelial cells, which may induce mucosal inflammatory response and subsequent damage to the surrounding tissue. The production and secretion of toxins into surrounding tissues by the proliferating fungus is re-
garded as an important virulence attribute and may facilitate fungal growth in the lung. Gliotoxin is the main toxin produced by A. fumigatus, others include helvolic acid, fumigatin and fumagillin. It is believed that toxins play a significant role in facilitating the colonization of the lung by A. fumigatus, as they can act to retard elements of the local immunity. Significantly, Aspergillus species that do not produce toxins to the same extent as A. fumigatus isolates are rarely seen in clinical cases. 7.3 Histoplasma capsulatum Histoplasma capsulatum is a dimorphic fungus that is the cause of histoplasmosis — the most prevalent fungal pulmonary infection in the USA. Histoplasmosis is common among AIDS patients but is also found among infants and geriatric subjects living in endemic areas who may be susceptible to infection because of impaired immune function. The mortality rate among infants exposed to a large dose of H. capsulatum may be 40–50%. H. capsulatum grows in the mycelial form in soil while in tissue it is encountered as round budding cells. Its natural habitat is soil that has been enriched with the droppings of bats or birds, and disturbance of such soil by natural (e.g. wind) or human (e.g. agriculture) activities releases large numbers of airborne spores that upon inhalation can establish pulmonary infection in individuals or in populations distant from the original source of spores. After inhalation of H. capsulatum, pulmonary macrophages engulf the yeast cells and provide a protected environment for the yeast to multiply and disseminate from the lungs to other tissues. The ability to survive within the hostile environment provided by the macrophage is the key to the success of H. capsulatum as a pathogen. Normal individuals who inhale a large number of H. capsulatum spores can develop a non-specific flulike illness associated with fever, chills, headache and chest pains after a 3-week incubation period that resolves without treatment. Chronic pulmonary histoplasmosis is seen in males in the 45–55-year age group who may have been exposed to low levels of spores over a long period of time. The condition is progressive, leads to a loss of lung function and death if untreated. 55
Chapter 4
7.4 Cryptococcus neoformans Cryptococcus neoformans is a capsulate yeast that is most frequently associated with infection in immunocompromised patients, particularly those with AIDS, where meningitis is the most common clinical manifestation. C. neoformans is a facultative intracellular pathogen that is capable of surviving and replicating within macrophages and withstanding the lytic activity within these cells. In order to survive within macrophages C. neoformans appears to accumulate polysaccharides within a cytoplasmic vesicle. As part of its survival strategy C. neoformans also produces melanin, which has the ability to bind and protect against microbicidal peptides. In addition, melaninization may allow the cell to withstand the effects of harmful hydroxyl radicals and so survive in hostile environments. The capsule of C. neoformans is a virulence factor in that it protects the cell from the immune response and capsular material (glucuronoxylomannans) induces the shedding of host cell adherence molecules required for the migration of host inflammatory cells to the site of infection, thus explaining the reduced inflammatory response to this yeast. In immunocompromised patients pulmonary infection can lead to disseminated forms of the disease where the eyes, skin and bones become infected. Cryptococcal meningitis is particularly associated with AIDS patients, where it is a major cause of death. While cryptococcosis may be controlled by antifungal therapy, in AIDS patients there is a danger of relapse unless antifungal therapy is constantly maintained. 7.5 Dermatophytes The dermatophytes are a group of keratinophilic fungi that can metabolize keratin — the principal protein in skin, nails and hair. Tinea capitis is defined as the infection of the hair and scalp with a dermatophyte, usually Microsporum canis or Trichophyton violaceum. In Europe and North Africa T. violaceum may be responsible for approximately 60% of cases of tinea capitis. This condition is characterized by a mild scaling and loss of hair and in some cases it may be contagious. Tinea corporis is 56
characterized by infection of the skin of the trunk, leg and arms with a dermatophyte and is usually caused by Trichophyton spp., Microsporum spp. or Epidermophyton floccosum. Lesions are itchy, dry and show scaling. Typically lesions retain a circular morphology and the condition is often referred to as ‘ringworm’. Tinea cruris is seen where the skin of the groin is infected with a dermatophytic fungus, usually E. floccosum or Trichophyton rubrum. This condition is highly contagious via fomites (towels, sheets, etc.) and up to 25% of patients show recurrence following antifungal therapy. Tinea pedis presents as infection of the feet with a dermatophyte and is very common and easily contracted. The principal fungi responsible for this condition are T. rubrum and E. floccosum and the sites of infection may include the webs of the toes, the sides of the feet or the soles of the feet. Tinea manuum is a fungal infection of the hands and can be caused by a range of fungi, most notably Trichophyton mentagrophytes. Infection is often seen in association with eczema and can result from transmission of fungi from another infected body site. Tinea unguium is defined as infection of the fingernails or toenails and is often described as onychomycosis, which includes infections due to a range of other fungi, bacteria and nail damage associated with certain disease states (e.g. psoriasis).
8 Antibiotic production by fungi Perhaps one of the most important discoveries regarding the beneficial use of fungi for humans was the identification in 1929 by Sir Alexander Fleming that an isolate of Penicillium notatum produced a substance capable of killing Gram-positive bacteria. This compound was subsequently identified as penicillin and was the first member of the b-lactam class of antibiotics to be discovered. These compounds function by inhibiting peptidoglycan synthesis in bacteria and their use has reduced the importance of the Gram-positive bacteria as a cause of disease. Subsequent to the identification of penicillin production by P. notatum a screen revealed that Penicillium chrysogenum was a superior producer. Following a series of mutagenic and selection procedures the strain used in conventional fermen-
Fungi
tations is capable of producing penicillin at a rate of 7000 mg/L compared with the 3 mg/L of Fleming’s P. notatum isolate. A typical penicillin fermentation yields three types of penicillin, namely F, G and V. The latter can be used directly; however, penicillin G is modified by the action of penicillin acylase to give a variety of semi-synthetic penicillins that show resistance to the action of bacterial penicillinases, which are implicated in conferring antibacterial drug resistance. The majority of antibiotics obtained from fungi are produced by fermentation and most are secondary metabolites, production of which occurs in the stationary phase and is linked to sporulation. Catabolite repression can inhibit antibiotic production and one way to avoid this is to use low levels of glucose in the fermentation medium or to obtain a mutant that is not catabolite-repressed. The chemical content of the medium must be monitored, as high levels of nitrogen or phosphate (PO4) retard antibiotic production. One problem that seriously affects the productivity of antibiotic fermentations is feedback inhibition, where the antibiotic builds to high intracellular levels and retards production or kills the cell. One means of reversing this is to introduce low levels of the antifungal agent amphotericin B, which increases membrane permeability, leading to a decrease in intracellular antibiotic levels and a concomitant increase in production. Antibiotic production can be maximized by optimizing production as a result of random mutagenesis and selection. Another approach has been to fuse or mate high producing strains with good secretors. Rational selection is a process where a chelating agent is introduced into the fermentation to complex all the metal ions present and consequently has a beneficial effect on antibiotic production. More recently genetic manipulation has been employed to express the genes for antibiotic production in another species that has the possibility of producing hybrid antibiotics with novel targets.
9 Genetic manipulation of fungi Fungi have been utilized for an extensive range of biotechnological applications. The S. cerevisiae expression system for producing recombinant protein
has a number of advantages including producing yields of purified secreted protein in the region of 20 mg/L. The yeast has been employed for the production of small hepatitis B surface proteins for use in recombinant hepatitis B vaccines and in the production of several recombinant malarial proteins, again for use in a vaccine. Antimalarial vaccine components produced by S. cerevisiae are currently in phase one clinical trials. S. cerevisiae has also been employed to produce the interferon class of cytokines, which are important in the treatment of diseases such as AIDS-associated Kaposi’s sarcoma, and to prevent re-occurrence of infections in patients suffering from chronic granulomatous disease. Yeasts are a popular choice for foreign gene expression as they have traditionally been classed as GRAS (generally regarded as safe), are easy to cultivate and are physically robust. From a genetic point of view S. cerevisiae is a eukaryotic organism, possesses a small genome and often contains natural plasmids (e.g. 2 mm circle) which can be utilized for transformation. In terms of their use as a molecular biological tool yeast strains can be transformed easily, are generally good secretors of protein, splice introns from animal genes and their RNA polymerase recognizes animal promoters. A number of vectors are utilized for transforming yeast cells and include integrative plasmids (e.g. Yip), independently replicating plasmids (e.g. Yrp) and specialized plasmids (e.g. yeast killer plasmid). Once a yeast has been transformed with a plasmid containing the gene of interest, the gene is transcribed and translated. Post-translation modification (e.g. glycosylation) occurs and secretion involves export of the protein of interest from the cell. As S. cerevisiae is not a prolific secretor of foreign protein, superior yeast secretors such as the yeast Candida maltosa, Yarrowia lipolytica and Pichia species have become more routinely employed in recent years. The ability to enzymically remove fungal cell walls and produce a protoplast has been exploited to generate strains capable of enhanced ethanol production or the metabolism of novel combinations of carbohydrates. Once protoplasts are formed they may be fused by incubation in polyethylene glycol (PEG) and calcium ions, which induces membrane breakdown. Selection of the resulting 57
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fusants ensures the isolation of hybrids combining characteristics of both parents. Protoplast fusion has been particularly successful in overcoming sexual incompatibility barriers that exist between fungal species. Protoplast fusion has been utilized to generate strains of S. cerevisiae that produce glucoamylase, overproduce the amino acid methionine and ferment xylose to ethanol. Fungal cell transformation is also possible once protoplasts have been isolated. Fungi have been utilized for thousands of years for the production of various foods and beverages. While these applications are still important, fungi are now being used in novel ways for the production of single cell protein, antibiotics, enzymes and as expression systems for the production and secretion of foreign proteins. Consequently, the continued use of fungi on a wide scale by humans is guaranteed.
10 Further reading Abu-Salah, K. (1996) Amphotericin B: an update. Br J Biomed Sci, 53, 122–133. Bennett, J. (1998) Mycotechnology: the role of fungi in biotechnology. J Biotechnol, 66, 101–107.
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Cohen, B. (1998) Amphotericin B toxicity and lethality: a tale of two channels. Int J Pharmaceut, 162, 95–106. Cotter, G. & Kavanagh, K. (2000) Adherence mechanisms of Candida albicans. Br J Biomed Sci, 57, 241–249. Daly, P. & Kavanagh, K. (2001) Pulmonary Aspergillosis: clinical presentation, diagnosis and therapy. Br J Biomed Sci, 58,197–205. Daum, G., Lees, N. D., Bard, M. & Dickson, R. (1998) Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast, 14, 1471–1510. Denning, D. (1996) Aspergillosis: diagnosis and treatment. Int J Antimicrob Agents, 6, 161–168. Jennings, D. & Lysek, G. (1999) Fungal Biology, 2nd edn. Bios Scientific Publishers, Oxford. Kavanagh, K. & Whittaker, P. A. (1996) Application of protoplast fusion to the non-conventional yeasts. Enz Microbiol Technol, 18, 45–51. Koltin, Y. & Hitchcock, C. (1997) The search for new triazole antifungal agents. Curr Opin Chem Biol, 1, 176–182. Odds, F. (1996) Resistance of clinically important yeast to antifungal agents. Int J Antimicrob Agents, 6, 145–147. Richardson, M. & Johnson, E. (2000) The Pocket Guide to Fungal Infection. Blackwell Science, Oxford. Stewart, G. (1997) Genetic manipulation of brewer’s and distiller’s yeast strains. Food Sci Technol Today, 11, 181–182. Sheehan, D., Hitchcock, C. & Sibley, C. (1999) Current and emerging azole antifungal agents. Clin Microbiol Rev, 12, 40–79. Walker, G. (1998) Yeast Physiology and Biotechnology. Wiley, Chichester, UK.
Chapter 5
Viruses Jean-Yves Maillard and David Stickler 1 Introduction 2 General properties of viruses 2.1 Size 2.2 Nucleic acid content 2.3 Metabolic capabilities 3 Structure of viruses 3.1 Helical symmetry 3.2 Icosahedral symmetry 4 The effect of chemical and physical agents on viruses 5 Virus–host cell interactions 6 Bacteriophages 6.1 The lytic growth cycle 6.2 Lysogeny 6.3 Epidemiological uses 7 Human viruses 7.1 Cultivation of human viruses
1 Introduction Following the demonstration by Koch and his colleagues that anthrax, tuberculosis and diphtheria were caused by bacteria, it was thought that similar organisms would, in time, be shown to be responsible for all infectious diseases. It gradually became obvious, however, that for a number of important diseases no such bacterial cause could be established. Infectious material from a case of rabies, for example, could be passed through special filters which held back all particles of bacterial size, and the resulting bacteria-free filtrate still proved to be capable of inducing rabies when inoculated into a susceptible animal. The term virus had, up until this time, been used quite indiscriminately to describe any agent capable of producing disease, so these filter-passing agents were originally called filterable viruses. With the passage of time the description ‘filterable’ has been dropped and the name virus has come to refer specifically to what are now known to be a distinctive group of microorganisms different
8
9 10 11 12 13
7.1.1 Cell culture 7.1.2 The chick embryo 7.1.3 Animal inoculation Multiplication of human viruses 8.1 Attachment to the host cell 8.2 Penetration of the viral particle 8.3 Uncoating of the viral particle 8.4 Replication of viral nucleic acids and translation of the genome 8.5 Maturation or assembly of virions 8.6 Release of virions into the surrounding environment The problems of viral chemotherapy Tumour viruses The human immunodeficiency virus Prions Further reading
in structure and method of replication from all others.
2 General properties of viruses All forms of life — animal, plant and even bacterial — are susceptible to infection by viruses. Three main properties distinguish viruses from their various host cells: size, nucleic acid content and metabolic capabilities. 2.1 Size Whereas a bacterial cell like a staphylococcus might be 1000 nm in diameter, the largest of the human pathogenic viruses, the poxviruses, measure only 250 nm along their longest axis, and the smallest, the poliovirus, is only 28 nm in diameter. They are mostly, therefore, beyond the limit of resolution of the light microscope and have to be visualized with the electron microscope. 59
Chapter 5
2.2 Nucleic acid content Viruses contain only a single type of nucleic acid, either DNA or RNA. 2.3 Metabolic capabilities Virus particles have no metabolic machinery of their own. They cannot synthesize their own protein and nucleic acid from inanimate laboratory media and thus fail to grow on even nutritious media. They are obligatory intracellular parasites, only growing within other living cells whose energy and protein-producing systems they redirect for the purpose of manufacturing new viral components. The production of new virus particles generally results in death of the host cell and as the particles spread from cell to cell (e.g. within a tissue), disease can become apparent in the host.
3 Structure of viruses In essence, virus particles are composed of a core of genetic material, either DNA or RNA, surrounded by a coat of protein. The function of the coat is to protect the viral genes from inactivation by adverse environmental factors, such as tissue nuclease enzymes which would otherwise digest a naked viral chromosome during its passage from cell to cell within a host. In a number of viruses the coat also plays an important part in the attachment of the virus to receptors on susceptible cells, and in many bacterial viruses the coat is further modified to facilitate the insertion of the viral genome through the tough structural barrier of the bacterial cell wall. The morphology of a variety of viruses is illustrated in Fig. 5.1. The viral protein coat, or capsid, is composed of a large number of subunits, the capsomeres. This subunit structure is a fundamental property and is important from a number of aspects. 1 It leads to considerable economy of genetic information. This can be illustrated by considering some of the smaller viruses, which might, for example, have as a genome a single strand of RNA composed of about 3000 nucleotides and a protein coat with an overall composition of some 20 000 amino acid 60
units. Assuming that one amino acid is coded for by a triplet of nucleotides, such a coat in the form of a single large protein would require a gene some 60 000 nucleotides in length. If, however, the viral coat comprised repeating units each composed of about 100 amino acids, only a section of about 300 nucleotides long would be required to specify the capsid protein, leaving genetic capacity for other essential functions. 2 Such a subunit structure permits the construction of the virus particles by a process in which the subunits self-assemble into structures held together by non-covalent intermolecular forces as occurs in the process of crystallization. This eliminates the need for a sequence of enzyme-catalysed reactions for coat synthesis. It also provides an automatic quality control system, as subunits which may have major structural defects fail to become incorporated into complete particles. 3 The subunit composition is such that the intracellular release of the viral genome from its coat involves only the dissociation of non-covalently bonded subunits, rather than the degradation of an integral protein sheath. In addition to the protein coat, many animal virus particles are surrounded by a lipoprotein envelope which has generally been derived from the cytoplasmic membrane of their last host cell. The geometry of the capsomeres results in their assembly into particles exhibiting one of two different architectural styles — helical or icosahedral symmetry (Fig. 5.2). There is a third structural group comprising the poxviruses and many bacterial viruses, in which a number of major structural components can be identified and the overall geometry of the particles is complex. 3.1 Helical symmetry Some virus particles have their protein subunits symmetrically packed in a helical array, forming hollow cylinders. The tobacco mosaic virus (TMV) is the classic example. X-ray diffraction data and electron micrographs have revealed that 16 subunits per turn of the helix project from a central axial hole that runs the length of the particle. The nucleic acid does not lie in this hole, but is
Viruses
Fig. 5.1 The morphology of a variety of virus particles. The large circle indicates the relative size of a staphylococcus cell.
embedded into ridges on the inside of each subunit and describes its own helix from one end of the particle to the other. Helical symmetry was thought at one time to exist only in plant viruses. It is now known, however, to occur in a number of animal virus particles. The influenza and mumps viruses, for example, which were first seen in early electron micrographs as roughly spherical particles, have now been observed as enveloped particles; within the envelope, the capsids themselves are helically symmetrical and appear similar to the rods of TMV, except that they are more flexible and are wound like coils of rope in the centre of the particle.
3.2 Icosahedral symmetry The viruses in this architectural group have their capsomeres arranged in the form of regular icosahedra, i.e. polygons having 12 vertices, 20 faces and 30 sides. At each of the 12 vertices or corners of these icosahedral particles is a capsomere, called a penton, which is surrounded by five neighbouring units. Each of the 20 triangular faces contains an identical number of capsomeres which are surrounded by six neighbours and called hexons. In plant and bacterial viruses exhibiting this type of symmetry, the hexons and pentons are composed of the same polypeptide chains; in animal viruses, 61
Chapter 5
however, they may be distinct proteins. The number of hexons per capsid varies considerably in different viruses. Adenovirus, for example, is constructed from 240 hexons and 12 pentons, while the much smaller poliovirus is composed of 20 hexons and 12 pentons.
Fig. 5.2 Icosahedral and helical symmetry in viruses.
4 The effect of chemical and physical agents on viruses Viruses generally exhibit greater sensitivity to chemical agents (biocides) than that shown by spore-forming organisms, but they may be more resistant than many species of bacteria, fungi and protozoa that do not form spores or cysts. Viruses can be divided into two major groups depending upon their sensitivity to biocides: the enveloped and the non-enveloped (naked) viruses. Enveloped viruses are generally large, and the presence of the lipid-containing envelope derived from the host cell enhances their susceptibility to chemical agents. Among naked viruses, the small ones such as picornaviruses (e.g. poliovirus) are among the most resistant to disinfection. Not all biocides have virucidal activity and the activity they do exhibit is subject to the environmental influences described in Chapter 11. Biocides probably have multiple target sites on viral particles and the overall damage caused results in loss of viral infectivity (Table 5.1). When compared to bacterial cells, however, viruses present only a few structural targets to biocides: the envelope (when present), the glycoproteins, the capsid and the nucleic acid. The activity of biocides against the viral envelope has not been well documented but it can be expected that membrane-active agents such as phenolics and the cationic biocides (e.g. chlorhexidine and quaternary ammonium compounds — QACs) will act against the viral envelope, which is a typical unit membrane. The capsid is
Table 5.1 Possible virus–biocide interactions Interaction
Consequence
Non-target sites attacked Reversible adsorption of biocides to anti-receptors
Virus inactivation Virus inactivation via loss of infectivity Reversible with the removal of the selective pressure (e.g. use of neutralizers) Virus inactivation via loss of infectivity Virus inactivation via loss of infectivity Reversible with the removal of the selective pressure Inactivation of the virus Release of possible intact and infectious viral nucleic acid Virus inactivation Complete virus inactivation
Non-reversible adsorption to anti-receptors Reversible conformational change of virus Destruction of the capsid Destruction of the nucleic acid within an intact capsid Destruction of the capsid and the viral nucleic acid
62
Viruses
probably the most important target site and any agents reacting strongly with protein amino groups (e.g. glutaraldehyde, ethylene oxide) or thiol groups (e.g. hypochlorite, iodine, ethylene oxide and hydrogen peroxide) may show virucidal activity. The release of an intact viral genome from a damaged capsid might be a cause for concern. The nucleic acid is the infectious part of the virus and its alteration/destruction by a biocide will result in viral inactivation. The size and nature of the genome and its interaction with the capsid (e.g. helical symmetry) certainly play a role in the sensitivity of the virus to biocides, and an increase in biocide concentration might result in an increase in the overall damage caused to a viral particle. In general, highly reactive biocides such as alkylating (e.g. glutaraldehyde) and oxidizing agents (e.g. peracetic acid) (Chapter 17) have good virucidal activity. However, other agents such as cationic biocides, phenolics and alcohols might have activity against enveloped viruses but a more limited effect on non-enveloped ones. Physical agents such as heat and irradiation can inactivate viruses, and these processes, together with good hygiene and active immunization (Chapters 8, 9 and 23), are of vital importance in the control of human viral diseases. Most viruses are readily inactivated following exposure at 60°C for 30 minutes and thermal processes are used for eliminating viruses (e.g. HIV) from blood products. However, some species are more resistant, and hepatitis B for example can survive long periods of exposure to higher temperatures, probably because of the number of viral particles present at one time. Viruses can withstand low temperatures and are routinely stored at -40°C to -70°C. The enveloped viruses are extremely sensitive to drying on surfaces, whereas much longer survival times are seen with non-enveloped viruses. UV irradiation can be used to eliminate viruses, especially airborne particles, by damaging viral nucleic acid, although again, small non-enveloped particles might be more resistant. Virus destruction can also be achieved by exposure to ionizing radiation (e.g. g-rays, accelerated electrons), which is used in terminal sterilization processes applied to pharmaceutical and medical products (Chapter 20).
Because of their small size many species of virus can enter the pores of filter membranes used for sterilization of liquids, but particle entrapment by the membrane is often achieved as a result of adsorption processes. As a consequence, substantial reductions in viral concentration can result from filtration through membranes of appropriate characteristics, and adequate assurance of sterility may be achieved provided that the pre-sterilization bioburden is carefully controlled (Chapter 20).
5 Virus–host cell interactions The precise sequence of events resulting from the infection of a cell by a virus will vary with different virus–host systems, but they will be variations of four basic themes. 1 Multiplication of the virus and destruction of the host cell. 2 Elimination of the virus from the cell and the infection aborted without a recognizable effect on the cells occurring. 3 Survival of the infected cell unchanged, except that it now carries the virus in a latent state. 4 Survival of the infected cell in a dramatically altered or transformed state, e.g. transformation of a normal cell to one having the properties of a cancerous cell.
6 Bacteriophages Bacteriophages, or as they are more simply termed, phages, are viruses that have bacteria as their host cells. The name was first given by D’Herelle to an agent which he found could produce lysis of the dysentery bacillus Shigella shiga. D’Herelle was convinced that he had stumbled across an agent with tremendous medical potential. His phage could destroy Sh. shiga in broth culture so why not in the dysenteric gut of humans? Similar agents were found before long which were active against the bacteria of many other diseases, including anthrax, scarlet fever, cholera and diphtheria, and attempts were made to use them to treat these diseases. It was a great disappointment, however, that phages so virulent in their antibacterial activity 63
Chapter 5
Fig. 5.3 T-even phage structure before and after tail contraction.
in vitro proved impotent in vivo. A possible exception was cholera, where some success seems to have been achieved, and cholera phages were apparently used by the medical corps of the German and Japanese armies during World War II to treat this disease. Since the development of antibiotics, however, phage therapy has been abandoned. Interest in bacterial viruses did not cease with the demise of phage therapy. They proved to be very much easier to handle in the laboratory than other viruses and had conveniently rapid multiplication cycles. They have, therefore, been used extensively as the experimental models for elucidating the biochemical mechanisms of viral replication. A vast amount of information has been collected about them and many of the important advances in molecular biology, such as the discovery of messenger RNA (mRNA), the understanding of the genetic code and the way in which genes are controlled, have come from work on phage–bacterium systems. It is probable that all species of bacteria are susceptible to phages. Any particular phage will exhibit a marked specificity in selecting host cells, attacking only organisms belonging to a single species. A Staphylococcus aureus phage, for example, will not infect Staph. epidermidis cells. In most cases, phages are in fact strain-specific, only being active on certain characteristic strains of a given species. Most phages are tadpole-shaped structures with heads which function as containers for the nucleic 64
acid and tails which are used to attach the virus to its host cell. There are, however, some simple icosahedral phages and others that are helically symmetrical cylinders. The dimensions of the phage heads vary from the large T-even group (Fig. 5.3) of Escherichia coli phages (60 ¥ 90 nm) to the much smaller ones (30 ¥ 30 nm) of certain Bacillus phages. The tails vary in length from 15 to 200 nm and can be quite complex structures (Fig. 5.3). While the majority of phages have double-stranded DNA as their genetic material, some of the very small icosahedral and the helical phages have single-stranded DNA or RNA. On the basis of the response they produce in their host cells, phages can be classified as virulent or temperate. Infection of a sensitive bacterium with a virulent phage results in the replication of the virus, lysis of the cell and release of new infectious progeny phage particles. Temperate phages can produce this lytic response, but they are also capable of a symbiotic response in which the invading viral genome does not take over the direction of cellular activity, the cell survives the infection and the viral nucleic acid becomes incorporated into the bacterial chromosome, where it is termed prophage. Cells carrying viral genes in this way are referred to as lysogenic. 6.1 The lytic growth cycle The replication of virulent phage was initially studied using the T-even-numbered (T2, T4, and T6)
Viruses
phages of E. coli. These phages adsorb, by their long tail fibres, on to specific receptors on the surface of the bacterial cell wall. The base plate of the tail sheath and its pins then lock the phage into position on the outside of the cell. At this stage, the tail sheath contracts towards the head, while the base plate remains in contact with the cell wall and, as a result, the hollow tail core is exposed and driven through to the cytoplasmic membrane (Fig. 5.3). Simultaneously, the DNA passes from the head, through the hollow tail core and is deposited on the outer surface of the cytoplasmic membrane, from where it finds its own way into the cytoplasm. The phage protein coat remains on the outside of the cell and plays no further part in the replication cycle. Within the first few minutes after infection, transcription of part of the viral genome produces ‘early’ mRNA molecules, which are translated into a set of ‘early’ proteins. These serve to switch off host cell macromolecular synthesis, degrade the host DNA and start to make components for viral DNA. Many of the early proteins duplicate enzymes already present in the host, concerned in the manufacture of nucleotides for cell DNA. However, the requirement for the production of 5hydroxymethylcytosine-containing nucleotides, which replace the normal cytosine derivatives in Teven phage DNA, means that some of the early enzymes are entirely new to the cell. With the build-up of its components, the viral DNA replicates and also starts to produce a batch of ‘late’ mRNA molecules, transcribed from genes which specify the proteins of the phage coat. These late messages are translated into the subunits of the capsid structures, which condense to form phage heads, tails and tail fibres, and then together with viral DNA are assembled into complete infectious particles. The enzyme digesting the cell wall, lysozyme, is also produced in the cell at this stage and it eventually brings about the lysis of the cell and liberation of about 100 progeny viruses, some 25 minutes after infection. As other phage systems have been studied, it has become clear that the T-even model of virulent phage replication is atypical in a number of respects. The large T-even genomes, with their coding capacity for about 200 proteins, give these phages a relatively high degree of independence from their hosts. Although relying on the host energy and
protein-synthesizing systems they are capable of specifying a battery of their own enzymes. Most other phages have considerably smaller genomes. They tend to disturb the host cell metabolism to a much lesser extent than the T-even viruses, and also rely to a greater degree on pre-existing cell enzymes to produce components for their nucleic acid. The lytic activity of the virulent phages can be demonstrated by mixing phage with about 107 sensitive indicator bacteria in 5 ml of molten nutrient agar. The mixture is then poured over the surface of a solid nutrient agar plate. On incubation, the phage particles will infect bacteria in their immediate neighbourhood, lysing them and producing a burst of progeny viruses. These particles then infect bacteria in the vicinity, producing a second generation of progeny and this sequence is repeated many times. In the meantime the uninfected bacteria produce a thick carpet or lawn of growth over the agar. As the lawn develops, clear holes or ‘plaques’ become obvious in it at each site of virus multiplication (Fig. 5.4). As each of these plaques is initiated by a single phage particle, they provide a means for titrating phage preparations. 6.2 Lysogeny When a temperate phage is mixed with sensitive indicator bacteria and plated as described above, the
Fig. 5.4 Plaques formed by a phage on a plate seeded with Bacillus subtilis.
65
Chapter 5
Fig. 5.5 Scheme to illustrate the lytic and lysogenic responses of bacteriophages.
reaction at each focus of infection is generally a combination of lytic and lysogenic responses. Some bacteria will be lysed and produce phage, others will survive as lysogenic cells, and the plaque becomes visible as a partial area of clearing in the bacterial lawn. It is possible to pick off cells from the central areas of these plaques and demonstrate that they carry prophage. The phage lambda (l) of E. coli is the temperate phage that has been most extensively studied. When any particular strain of E. coli, say K12, is infected with l, the cells surviving the infection are designated E. coli K12(l) to indicate that they are carrying the l-prophage. The essential features of lysogenic cells and the phenomenon of lysogeny are listed below and summarized in Fig. 5.5. 1 Integration of the prophage into the bacterial chromosome ensures that, on cell division, each daughter cell will acquire the set of viral genes. 2 In a normally growing culture of lysogenic bacteria, the majority of bacteria manage to keep 66
their prophages in a dormant state. In a very small minority of cells, however, the prophage genes express themselves. This results in the multiplication of the virus, lysis of the cells and liberation of infectious particles into the medium. 3 Exposure of lysogenic cultures to certain chemical and physical agents, e.g. hydrogen peroxide, mitomycin C and ultraviolet light, results in mass lysis and the production of high titres of phage. This process is called induction. 4 When a lysogenic cell is infected by the same type of phage as it carries as prophage, the infection is aborted, the activity of the invading viral genes being repressed by the same mechanism that normally keeps the prophage in a dormant state. 5 Lysogeny is generally a very stable state, but occasionally a cell will lose its prophage and these ‘cured’ cells are once more susceptible to infection by that particular phage type. Lysogeny is an extremely common phenomenon and it seems that most natural isolates of bacteria carry one or more prophages: some strains of Staph.
Viruses
aureus have been shown to carry four or five different prophages. The induction of a lysogenic culture to produce infectious phages, followed by lysogenization of a second strain of the bacterial species by these phages, results in the transmission of a prophage from the chromosome of one type of cell to that of another. On this migration, temperate bacteriophages can occasionally act as vectors for the transfer of bacterial genes between cells. This process is called transduction and it can be responsible for the transfer of such genetic factors as those that determine resistance to antibiotics (Chapter 13). In addition, certain phages have the innate ability to change the properties of their host cell. The classic example is the case of the b-phage of Corynebacterium diphtheriae. The acquisition of the bprophage by non-toxin-producing strains of this species results in their conversion to diphtheria toxin-producers. 6.3 Epidemiological uses Different strains of a number of bacterial species can be distinguished by their sensitivity to a collection of phages. Bacteria which can be typed in this way include Staph. aureus and Salmonella typhi. The particular strain of, say, Staph. aureus responsible for an outbreak of infection is characterized by the pattern of its sensitivity to a standard set of phages and then possible sources of infection are examined for the presence of that same phage type of Staph. aureus. More recently, the fact that many of the chemical agents which cause the induction of prophage are carcinogenic has led to the use of lysogenic bacteria in screening tests for detecting potential carcinogens.
7 Human viruses Viruses are, of course, important and common causes of disease in humans, particularly in children. Fortunately, most infections are not serious and, like the rhinovirus infections responsible for the common cold syndrome, are followed by the complete recovery of the patient. Many viral infec-
tions are in fact so mild that they are termed ‘silent’, to indicate that the virus replicates in the body without producing symptoms of disease. Occasionally, however, some of the viruses that are normally responsible for mild infections can produce serious disease. This pattern of pathogenicity is exemplified by the enterovirus group. Most enterovirus infections merely result in the symptomless replication of the virus in the cells lining the alimentary tract. Only in a small percentage of infections does the virus spread from this site via the bloodstream and the lymphatic system to other organs, producing a fever and possibly a skin rash in the host. On rare occasions enteroviruses like poliovirus can progress to the central nervous system where they may produce an aseptic meningitis or paralysis. There are a few virus diseases, such as rabies, which are invariably severe and have very high mortality rates. Human viruses will cause disease in other animals. Some are capable of infecting only a few closely related primate species, others will infect a wide range of mammals. Under the conditions of natural infection viruses generally exhibit a considerable degree of tissue specificity. The influenza virus, for example, replicates only in the cells lining the upper respiratory tract. Table 5.2 presents a summary of the properties of some of the more important human viruses. 7.1 Cultivation of human viruses The cultivation of viruses from material taken from lesions is an important step in the diagnosis of many viral diseases. Studies of the basic biology and multiplication processes of human viruses also require that they are grown in the laboratory under experimental conditions. Human pathogenic viruses can be propagated in three types of cell systems. 7.1.1 Cell culture Cells from human or other primate sources are obtained from an intact tissue, e.g. human embryo kidney or monkey kidney. The cells are dispersed by digestion with trypsin and the resulting suspension of single cells is generally allowed to settle in a vessel containing a nutrient medium. The cells will metabolize and grow and after a few days of incubation at 67
Chapter 5 Table 5.2 Important human viruses and their properties Group
Virus
Characteristics
Clinical importance
Variola Vaccinia
Large particles 200– 250 nm: complex symmetry
Variola is the smallpox virus, it produces a systemic infection with a characteristic vesicular rash affecting the face, arms and legs, and has a high mortality rate. Vaccinia has been derived from the cowpox virus and is used to immunize against smallpox
Adenoviruses
Adenovirus
Icosahedral particles 80 nm in diameter
Commonly cause upper respiratory tract infections; tend to produce latent infections in tonsils and adenoids; will produce tumours on injection into hamsters, rats or mice
Herpesviruses
Herpes simplex virus (HSV1 and HSV2)
Enveloped, icosahedral particles 150 nm in diameter
HSV1 infects oral membranes in children, >80% are infected by adolescence. Following the primary infection the individual retains the HSV1 DNA in the trigeminal nerve ganglion for life and has a 50% chance of developing ‘cold sores’. HSV2 is responsible for recurrent genital herpes
Varicella-zoster virus (VZV)
Enveloped, isocahedral particles 150 nm in diameter
Causes chickenpox in children; virus remains dormant in any dorsal root ganglion of the CNS; release of immune control in the elderly stimulates reactivation resulting in shingles
Cytomegalovirus (CMV)
Enveloped, icosahedral particles 150 nm in diameter
CMV is generally acquired in childhood as a subclinical infection. About 50% of adults carry the virus in a dormant state in white blood cells. The virus can cause severe disease (pneumonia, hepatitis, encephalitis) in immunocompromised patients. Primary infections during pregnancy can induce serious congenital abnormalities in the fetus
Epstein–Barr virus (EBV)
Enveloped, icosahedral particles 150 nm in diameter
Infections occur by salivary exchange. In young children they are commonly asymptomatic but the virus persists in a latent form in lymphocytes. Infection delayed until adolescence often results in glandular fever. In tropical Africa, a severe EBV infection early in life predisposes the child to malignant facial tumours (Burkitt’s lymphoma)
Hepatitis viruses
Hepatitis B virus (HBV)
Spherical enveloped particle 42 nm in diameter enclosing an inner icosahedral 27-nm nucleocapsid
In areas such as South-East Asia and Africa, most children are infected by perinatal transmission. In the Western world the virus is spread through contact with contaminated blood or by sexual intercourse. There is strong evidence that chronic infections with HBV can progress to liver cancer
Papovaviruses
Papilloma virus
Naked icosahedra 50 nm in diameter
Multiply only in epithelial cells of skin and mucous membranes causing warts. There is evidence that some types are associated with cervical carcinoma
RNA viruses Myxoviruses
Influenza virus
Enveloped particles, 100 nm in diameter with a helically symmetric
These viruses are capable of extensive antigenic variation, producing new types against which the human population does not have effective immunity. These new antigenic
DNA viruses Poxviruses
68
Viruses Table 5.2 Continued Group
Characteristics
Clinical importance
capsid; haemagglutinin and neuraminidase spikes project from the envelope
types can cause pandemics of influenza. In natural infections the virus only multiplies in the cells lining the upper respiratory tract. The constitutional symptoms of influenza are probably brought about by absorption of toxic breakdown products from the dying cells on the respiratory epithelium
Mumps virus
Enveloped particles variable in size, 110–170 nm in diameter, with helical capsids
Infection in children produces characteristic swelling of parotid and submaxillary salivary glands. The disease can have neurological complications, e.g. meningitis, especially in adults
Measles virus
Enveloped particles variable in size, 120–250 nm in diameter, helical capsids
Very common childhood fever, immunity is life-long and second attacks are very rare
Rhabdoviruses
Rabies virus
Bullet-shaped particles, 75–180 nm, enveloped, helical capsids
The virus has a very wide host range, infecting all mammals so far tested; dogs, cats and cattle are particularly susceptible. The incubation period of rabies is extremely varied, ranging from 6 days up to 1 year. The virus remains localized at the wound site of entry for a while before passing along nerve fibres to central nervous system, where it invariably produces a fatal encephalitis
Reoviruses
Rotavirus
An inner core is surrounded by two concentric icosahedral shells producing particles 70 nm in diameter
A very common cause of gastroenteritis in infants. It is spread through poor water supplies and when standards of general hygiene are low. In developing countries it is responsible for about a million deaths each year
Picornaviruses
Poliovirus
Naked icosahedral particles 28 nm in diameter
One of a group of enteroviruses common in the gut of humans. The primary site of multiplication is the lymphoid tissue of the alimentary tract. Only rarely do they cause systemic infections or serious neurological conditions like encephalitis or poliomyelitis
Rhinoviruses
Naked icosahedra 30 nm in diameter
The common cold viruses; there are over 100 antigenically distinct types, hence the difficulty in preparing effective vaccines. The virus is shed copiously in watery nasal secretions
Hepatitis A virus (HAV)
Naked icosahedra 27 nm in diameter
Responsible for ‘infectious hepatitis’ spread by the oro-faecal route especially in children. Also associated with sewage contamination of food or water supplies
Rubella
Spherical particles 70 nm in diameter, a tightly adherent envelope surrounds an icosahedral capsid
Causes German measles in children. An infection contracted in the early stages of pregnancy can induce severe multiple congenital abnormalities, e.g. deafness, blindness, heart disease and mental retardation
Paramyxoviruses
Togaviruses
Virus
69
Chapter 5 Table 5.2 Continued Group
Virus
Characteristics
Clinical importance
Flaviviruses
Yellow fever virus
Spherical particles 40 nm in diameter with an inner core surrounded by an adherent lipid envelope
The virus is spread to humans by mosquito bites; the liver is the main target; necrosis of hepatocytes leads to jaundice and fever
Hepatitis C virus (HCV)
Spherical particles 40 nm in diameter consisting of an inner core surrounded by an adherent lipid envelope
The virus is spread through blood transfusions and blood products. Induces a hepatitis which is usually milder than that caused by HBV
Filoviruses
Ebola virus
Long filamentous rods composed of a lipid envelope surrounding a helical nucleocapsid 1000 nm long, 80 nm in diameter
The virus is widespread amongst populations of monkeys. It can be spread to humans by contact with body fluids from the primates. The resulting haemorrhagic fever has a 90% case fatality rate
Retroviruses
Human T-cell leukaemia virus (HTLV-1)
Spherical enveloped virus 100 nm in diameter, icosahedral cores contain two copies of linear RNA molecules and reverse transcriptase
HTLV is spread inside infected lymphocytes in blood, semen or breast milk. Most infections remain asymptomatic but after an incubation period of 10–40 years in about 2% of cases, adult T-cell leukaemia can result
Human Differs from other immunodeficiency retroviruses in that the virus (HIV) core is cone-shaped rather than icosahedral Hepatitis viruses
Hepatitis D virus (HDV)
An RNA-containing virus The presence of the satellite HDV exacerbates the that can only replicate in pathogenic effects of HBV producing severe hepatitis cells co-infected with HBV. The spherical coat of HDV is composed of HBV capsid protein
37°C will form a continuous film or monolayer one cell thick. These cells are then capable of supporting viral replication. Cell cultures may be divided into three types according to their history. 1 Primary cell cultures, which are prepared directly from tissues. 2 Secondary cell cultures, which can be prepared by taking cells from some types of primary culture, usually those derived from embryonic tissue, dispersing them by treatment with trypsin and inoculating some into a fresh batch of medium. A limited 70
HIV is transmitted from person to person via blood or genital secretions. The principal target for the virus is the CD4+ T-lymphocyte cells. Depletion of these cells induces immunodeficiency
number of subcultures can be performed with these sorts of cells, up to a maximum of about 50 before the cells degenerate. 3 There are now available a number of lines of cells, mainly originating from malignant tissue, which can be serially subcultured apparently indefinitely. These established cell lines are particularly convenient as they eliminate the requirement for fresh animal tissue for such sets or series of cultures. An example of these continuous cell lines are the famous HeLa cells, which were originally isolated
Viruses
Fig. 5.6 The cytopathic effect of a virus on a
tissue culture cell monolayer.
from a cervical carcinoma of a woman called Henrietta Lacks, long since dead but whose cells have been used in laboratories all over the world to grow viruses. Inoculation of cell cultures with virus-containing material produces characteristic changes in the cells. The replication of many types of viruses produces the cytopathic effect (CPE) in which cells degenerate. This effect is seen as the shrinkage or sometimes ballooning of cells and the disruption of the monolayer by death and detachment of the cells (Fig. 5.6). The replicating virus can then be identified by inoculating a series of cell cultures with mixtures of the virus and different known viral antisera. If the virus is the same as one of the types used to prepare the various antisera, then its activity will be neutralized by that particular antiserum and CPE will not be apparent in that tube. Alternatively viral antisera labelled with a fluorescent dye can be used to identify the virus in the cell culture.
viruses, for example, can be grown in the cells of the membrane bounding the amniotic cavity, while smallpox virus will grow in the chorioallantoic membrane. The growth of smallpox virus in the embryo is recognized by the formation of characteristic pock marks on the membrane. Influenza virus replication is detected by exploiting the ability of these particles to cause erythrocytes to clump together. Fluid from the amniotic cavity of the infected embryo is titrated for its haemagglutinating activity. 7.1.3 Animal inoculation Experimental animals such as mice and ferrets have to be used for the cultivation of some viruses. Growth of the virus is indicated by signs of disease or death of the inoculated animal.
8 Multiplication of human viruses 7.1.2 The chick embryo Fertile chicken eggs, 10–12 days old, have been used as a convenient cell system in which to grow a number of human pathogenic viruses. Figure 5.7 shows that viruses generally have preferences for particular tissues within the embryo. Influenza
The objective of viral replication is to ensure the multiplication of the virus by formation of virions (virus progeny) identical to the parent strain. Because of the structure of a virus, the multiplication cycle focuses mainly on the replication of viral DNA or RNA. 71
Chapter 5
Fig. 5.7 A chick embryo showing the inoculation routes for virus cultivation.
The progress made in cell culture techniques has provided a better understanding of viral replication cycles. Human viruses generally have a slow multiplication cycle requiring from 4 to more than 40 hours (in some herpesviruses) for completion; this contrasts with bacterial viruses (bacteriophages) with a replication cycle as fast as 30 minutes. Certain viruses exhibit low infectivity; for example, picornavirus infectivity can be as low as 0.1% and rotavirus about 0.2%, and this makes the study of viral replication difficult. In general, viral replication cycles, whatever the differences in detail, comprise events that can be separated into six discrete stages (Fig. 5.8): • Attachment to the host cell • Penetration of the host cell membrane • Uncoating of the virus to release its core components • Replication of the viral nucleic acids and translation of the genome • Maturation or re-assembly of virions (i.e. progeny virus particles) • Release of virions into the surrounding environment.
the viral envelope. These structures act as antireceptors that bind to a surface receptor on the target cells and hence they play an important role in the host–virus specificity, although some viruses may share common cell receptors. The virus–cell recognition event is similar to any protein–protein interaction in that it occurs through a stereospecific network of hydrogen bonds and lipophilic associations. Viral attachment to the cell surface can be divided into three phases: (i) an initial contact mainly dependent on Brownian motion, (ii) a reversible phase during which electrostatic repulsion is reduced and (iii) irreversible changes in antireceptor–receptor configuration that initiates viral penetration through the cell membrane. A classic example of this process is the haemagglutinin antireceptor of influenza virus. It binds the terminal glycoside residues of gangliosides (cell surface glycolipids); this leads directly to the virus particle adhering to the cell. A similar interaction occurs during the attachment of HIV virus particles to T lymphocytes (see later). 8.2 Penetration of the viral particle
8.1 Attachment to the host cell All viruses contain (glyco)protein materials on their surface, whether as an integral part of the capsid structure or in the form of spikes embedded in 72
Once irreversibly attached to the cell surface, configuration changes of the complex anti-receptor– receptor initiate the penetration of the viral particle through the cell membrane. This energy-dependent
Viruses
Host cell
Cell nucleus
Viral particle
maturation reassembly
Viral genome replication
Release of virions
Attachment Penetration Virus enclosed in a cytosolic vacuole
Virus enclosed in a cytosolic vacuole Viral enzymes
Structural viral proteins
Endosomes Uncoating Host polymerases and ribosomes
Host polymerases and ribosomes
Early mRNA and protein synthesis
Early mRNA and protein synthesis
Fig. 5.8 Diagrammatic representation of the production and release of viral particles from an infected cell.
step occurs rapidly and operates by two mechanisms: endocytosis and fusion. • During endocytosis, the association between receptor and anti-receptor draws the cell membrane to engulf the virus particle forming a cytosolic vacuole, similar to the process by which cells ingest other materials. Probably all non-enveloped viruses penetrate the cell in this manner, but some enveloped viruses such as orthomyxoviruses (e.g. influenza) also use this process. • Fusion of the viral envelope and the cell membrane results in the direct release of the capsid into the cell cytoplasm. Herpesviruses and HIV penetrate their host cells in this manner. The precise biophysical details of the fusion process remain unknown.
8.3 Uncoating of the viral particle Uncoating refers to the events after penetration which allow the virus to express its genome. For viruses that penetrate the cell via endocytosis, conformational changes in the virus coat and release of viral nucleocapsid into the cytoplasm result from acidification of the virus-containing cytosolic vacuoles following endosome fusion. Other viruses require only partial uncoating (e.g. reovirus) before transcription of their nucleic acid can begin, but in most cases the viral capsid completely disintegrates before viral functions start to be expressed. In some cases the nucleocapsid passes to the cell nucleus before uncoating occurs. The uncoating process is increasingly studied be73
Chapter 5
cause of its potential for antiviral chemotherapy. For example, amantadine and rimantadine (Fig. 5.9) are anti-influenza drugs, which function in part by inhibiting the envelope viral protein M2, resulting in the remaining association of the viral nucleocapsid with matrix proteins and preventing its transfer to the nucleus. 8.4 Replication of viral nucleic acids and translation of the genome This stage of the viral replication cycle mainly involves three basic biochemical processes: the synthesis of new viral proteins (translation of the genome), RNA synthesis (transcription) and viral genome replication. There is a tremendous diversity in the structure, size and nature of the nucleic acid of mammalian viruses. There is no single pattern of replication, but all viruses make proteins with function to (i) alter the host cell metabolism in favour of virion production, (ii) ensure the replication of the viral genome and (iii) package the genome into virus particles. Viral protein synthesis can be divided into early and late protein synthesis. Early synthesis is concerned with the production of proteins involved in the replication of the viral genome such as polymerases, while late synthesis is more concerned with the production of structural components of the new virions. The mRNA molecules are, of course, translated on the cytoplasmic ribosomes. The way in which the viral genome is replicated depends entirely on the nature of the nucleic acid carried by the virus. Positive strand RNA viruses (e.g. poliovirus) can use the parent RNA directly as mRNA, after the acquisition from the host cell of a terminal sequence enabling immediate translation. With negative strand RNA viruses (e.g. influenza virus), a positive RNA strand complementary in base sequence to the parent RNA has to be transcribed using an RNA-dependent RNA polymerase carried by the virus, as eukaryotic cells do not possess such enzymes. Retroviruses (e.g. HIV) produce a proviral DNA from their positive strand RNA using the chemotherapeutically important reverse transcriptase enzyme. This is a unique enzyme acting both as RNA- and DNA-directed DNA polymerase, and as 74
an associated RNAse activity. Its function is to produce a double-stranded proviral DNA, which is transported to the nucleus and integrated in the host genome under the control of a viral integrase. With the DNA-containing viruses, e.g. adenovirus, the nucleic acid passes to the nucleus where a host cell DNA-dependent RNA polymerase is used to transcribe part of the viral genome. The poxvirus, vaccinia, uses its own DNA-dependent RNA polymerase which is released during uncoating. For this virus the whole replication takes place in the cell cytoplasm, while for other DNA viruses, proteins synthesized in the cytoplasm are transported back to the nucleus where the virion assembly process takes place. Although the replication of the viral genome is mainly under the control of polymerases, some complex viruses code for regulatory factors which suppress or accelerate cellular processes according to the need of the virus (e.g. tat gene in HIV). The involvement of host cell enzymes in the replication of the viral proteins and genome severely limits the design of new antiviral drugs. However, unlike host mRNAs, which code directly for functional proteins, most viral genomes are polycistronic, which means that all the information is, or becomes, encoded in one piece of mRNA. As a result, the polyprotein produced by translation needs to be cleaved at appropriate positions for the different proteins to be functional. This post-translational protein processing offers targets for antiviral chemotherapy. Viruses that possess a polycistronic genome encode for a virus-specific protease, which is capable of performing cleavages at the correct place. Many of these viral proteases have been identified and characterized and have led to the discovery of several inhibitors, such as indinavir, sequinavir and ritonavir used for HIV chemotherapy. 8.5 Maturation or assembly of virions As large amounts of viral materials accumulate within the cell, the role of structural proteins becomes evident. The structural proteins of small non-enveloped viruses (e.g. poliovirus) selfassemble once they are synthesized. Initially individual units form capsomeres, which then associate into capsids. The viral genome and associated
Viruses
proteins necessary for viral replication become engulfed in this new structure. Most non-enveloped viruses accumulate within the cytoplasm or nucleus and are only released when the cell lyses. All enveloped human viruses acquire their phospholipid coating by budding through cellular membranes. For example, with the influenza virus, the capsid protein subunits are transported from the ribosomes to the nucleus, where they combine with new viral RNA molecules and are assembled into the helical capsids. The haemagglutinin and neuraminidase proteins that project from the envelope of the normal particles migrate to the cytoplasmic membrane where they displace the normal cell membrane proteins. The assembled nucleocapsids finally pass out from the nucleus, and as they impinge on the altered cytoplasmic membrane they cause it to bulge and bud off completed enveloped particles from the cell. Other enveloped viruses, such as herpesviruses, assemble within the nucleus and acquire their envelope as they pass through the inner nuclear membrane. From here they pass into a vesicle that protects the virions from the cytoplasm and migrates to the cell surface. The assembly of virions is not fully understood. It is thought that cellular factors such as chaperone proteins and the interactions between the viral nucleic acid and structural proteins might play an important role during the maturation process. 8.6 Release of virions into the surrounding environment The release of virions ends the replication cycle of a virus. Two main release routes can be distinguished: budding and lysis. Most non-enveloped viruses accumulate within the cytoplasm or the nucleus and are only released when the cell lyses. The cell often self-disintegrates because normal cell housekeeping function is not maintained during viral replication. It has also been suggested that some viral proteases trigger the cell cytoplasmic membrane disintegration. Enveloped viruses usually exit the cell by budding. Virus particles are released in this way over a period of hours before the cell eventually dies. In the case of influenza virus, neuraminidase catalyses the cleavage of terminal neuraminic acid residues from
cell surface gangliosides, which frees the virus particle from its interaction with the cell.
9 The problems of viral chemotherapy Vaccination programmes have been very effective in the control of some viral diseases. Protection against smallpox was first demonstrated in 1796 by Jenner by inoculation with cowpox. Smallpox was later successfully eradicated. Poliovirus is also being eliminated thanks to a controlled intensive worldwide vaccination programme initiated by the World Health Organization. Such preventative methods are the most effective and economic way of controlling viral infections. There are several possible biochemical target sites within the replication cycle of a virus (Table 5.3). The virus–receptor interaction, which is specific for the host cells (e.g. CD4–gp120 interaction between T lymphocytes and HIV), can potentially be blocked, although this approach would have a preventative rather than therapeutic role. There is greater potential for blocking the penetration and especially the uncoating processes. Some molecules (polypeptides) that bind to the viral coat are already being tested for their efficacy to block this viral replication step. However, the better prospects and most of the effective antiviral drugs available focus on the replication of viral core components, translation of the genome, and nucleic acid transcription and replication (e.g. DNA polymerase). Finally, it is possible that, to some extent, the maturation, assembly and release phase of virus particles could be blocked, although this process would be virusdependent. For example, two very potent and highly selective neuraminidase inhibitors, zanamivir and ozaltamivir have been approved for clinical use against the influenza virus. These inhibitors prevent the shedding of virions and the transmission of infection. With the increase in understanding of viral replication and in particular viral protein and nucleic acid synthesis, and the development of reliable and sophisticated antiviral assays, the young science of antiviral chemotherapy has progressed tremendously over the last 50 years. Acyclovir (Fig. 5.9) was probably the first truly effective and selective 75
Chapter 5 Table 5.3 Possible target sites for chemotherapeutic agents Target sites
Examples
Attachment, penetration and uncoating
Inhibition of HIV-1 attachment. Competition for CD4 receptors using a pentapeptide identical in sequence to the terminal amino acids of gp120* Inhibition of herpesvirus attachment. Inhibition of ribonucleotide reductase using a polypeptide* Inhibition of influenza virus attachment. Competition for cell receptor using a hexapeptide fusion sequence at the N-terminus of the haemagglutinin antireceptor* Inhibition of viral coded reverse transcriptase using nucleoside analogues (e.g. ddl, 3TC, d4T, AZT) Inhibition of viral coded reverse transcriptase using non-nucleoside analogues (e.g. foscarnet) Inhibition of intracellular processing of viral proteins (e.g. alteration of glycosylation)† Inhibition of viral DNA replication using nucleoside analogues (e.g. idoxuridine against herpesvirus) Use of oligonucleotides therapy (e.g. antisense nucleotides)† Use of nucleoside analogues such as acyclovir Foscarnet against herpesviruses Protease inhibitors such as indinavir, saquinavir (for HIV chemotherapy)
Gene transcription and genome replication
Inhibition of viral mRNA function Inhibition of viral DNA replication via the inhibition of specific polymerases Inhibition of low molecular weight polypeptide gene products
* Currently being studied and developed. † Target site being considered for antiviral design and development.
antiviral agent. Viral polymerases have recently been the most widely studied target for antiviral chemotherapy, given that the replication of a virus is blocked by selectively blocking the production of viral nucleic acids. Nucleoside analogues such as acyclovir inhibit viral nucleic acid polymerases; this is probably the single most important chemotherapeutic interaction to date. For HIV chemotherapy, the inhibition of the viral encoded reverse transcriptase constitutes a major target site. Several nucleoside analogues such as didanosine (ddl), lamivudine (3TC), stavudine (d4T) and zidovudine (ZDV, formerly azidothymidine — AZT; Fig 5.9) are widely available. Nucleoside analogues are also used to inhibit the replication of viral DNA. For example, idoxiuridine (trifuridine) is incorporated into viral and cellular DNA instead of thymidine and is used for the treatment of herpes simplex virus and cytomegalovirus infections. Other non-nucleoside analogues such as nevirapine and foscarnet (a phosphate analogue; Fig. 5.9) are also employed in therapy, and oligonucleotides (nucleic acid oligomers) 76
with base sequence complimentary to conserved regions of pro-viral DNA have been successful in the prevention of viral mRNA function. Oligonucleotide therapies can be divided into four separate approaches: • Antisense nucleotides: production of an RNA sequence complementary to single-stranded viral RNA, which triggers the formation of doublestranded duplex, inhibiting viral RNA replication. • Antigen methods: formation of triple helix of DNA preventing transcription. • Decoy methods: production of synthetic decoys corresponding to a specific nucleic acid sequence which binds virally encoded regulatory proteins and affects transcription. • Ribozymes: production of RNA molecules (oligo (ribo)nucleotides) inducing cleavage of other RNA sequences at specific sites. Finally, there is an increased use of protease inhibitors (e.g. indinavir sulphate) that inhibit viral low molecular weight polypeptide gene products. Despite the tremendous increase in antiviral
Viruses O N HO
O
O NH
N
OH
NH2
N
Acyclovir (nucleoside analogue of guanosine)
O
HO P
OH
Foscarnet (phosphate analogue; trisodium salt of phosphonoformic acid)
O O
NH HO
O
N
NH O HO
ON
O
N3 AZT (nucleoside analogue of thymidine)
d4T (nucleoside analogue of thymidine)
O N HO
Fig. 5.9 Examples of antiviral drugs.
O
N
NH2
NH2
NH N
ddl (nucleoside analogue of guanosine)
drugs, there are several problems inherent in antiviral chemotherapy: • Delay in treatment: many viral diseases only become apparent after extensive viral multiplication and tissue damage have occurred. • Drug toxicity (e.g. nucleoside analogues): viral replication has considerable dependence on host cell enzyme systems that produce energy and synthesize proteins or other biopolymers. Furthermore, such compounds can also interfere with host regulatory genes, triggering cell apoptosis. • Degradation and penetration of the antiviral drugs: the use of pro-drugs might improve drug absorption and other pharmacokinetics properties. For example, valacyclovir, a valyl ester of acyclovir is better absorbed into the gastrointestinal tract than the parent drug, and is cleaved by host enzymes into valine (a natural amino acid) and acyclovir. • Finally, some viruses have developed resistance to antiviral drugs. HIV chemotherapy now involves
CH2
Amantadine
Rimantadine
the use of at least two anti-HIV agents having different mechanisms of action (for example, zidovudine (AZT) + lamivudine (3TC) + indinavir (a protease inhibitor) or stavudine (d4T) + didanosine (ddl) ± indinavir), in order to minimize the development of HIV resistance to individual antiviral drugs, prolong the antiviral effect and improve outcome.
10 Tumour viruses Experimental infection of animals has shown that certain viruses can induce cancer. This oncogenic activity can also be demonstrated in vitro in cell cultures. Cells surviving viral infection can change dramatically, acquiring the characteristics of tumour cells. These transformed cells exhibit frequent mitosis. They also lose the property of cell contact inhibition, so they tend to pile up on top of each other rather than remaining as organized monolayers. 77
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Viral DNA can be recovered from cells transformed by DNA-containing viruses and in some cases these viral DNA sequences have been shown to be integrated into the host cell DNA. The oncogenic RNAcontaining viruses possess the enzyme reverse transcriptase, which makes DNA copies of the viral RNA genome, and cells transformed by these retroviruses contain these DNA transcripts integrated into host cell DNA. It appears that the acquisition of DNA from oncogenic viruses can disturb the systems that normally regulate cell growth and division. There is a now a substantial body of evidence that viruses are a major cause of cancer in humans, being involved in the genesis of some 20% of human cancers worldwide. It has also become clear, however, that less than 1% of individuals infected with these oncogenic viruses will develop cancer and that there may be intervals of many years before the cancer becomes apparent. There seems to be no single mechanism by which viruses induce tumours. Virus infection is one step in a multi-stage, multifactorial process. The acquisition of viral genes must be followed by other events such as environmental or dietary exposure to chemical carcinogens, or infection by other microorganisms if progression to cancer is to occur. The host genotype may also be a co-factor. The Epstein–Barr virus (EBV) is a herpesvirus that is associated with the formation of lymphomas and nasopharyngeal carcinomas. It infects most humans but infections in childhood are generally asymptomatic. In young adults it can cause glandular fever, a chronic condition in which there is proliferation of white blood cells. After infection the virus becomes latent in B lymphocytes for the lifetime of the individual. In vitro the infection of B lymphocytes by EBV results in their transformation and proliferation. Normally in vivo, this EBVinduced proliferation of B cells is kept under control by the action of T-killer lymphocytes. In some African children infection with EBV induces Burkitt’s lymphoma, a particularly malignant tumour of the jaw. The characteristic occurrence of this condition in hot humid regions of Africa where mosquitoes flourish has led to the hypothesis that infection with EBV has to be followed by malaria before the lymphomas will develop. Suppression of 78
T-cell activity is known to occur in malaria and it is thought that in this situation infection with the malarial parasite provides the co-factor that triggers tumour formation. Nasopharyngeal carcinoma is rare in the general human population. However, in certain parts of south China and in Cantonese populations that have emigrated to other parts of the world it is the most common form of cancer. Infection with EBV occurring at any age is the primary stage in the process. It then seems that the consumption of large amounts of smoked fish (a cultural characteristic of this group) exposes them to carcinogenic nitrosamines, which act as the cofactors to trigger cell proliferation. The hepatitis B virus (HBV) is strongly associated with liver cancer. Studies have shown that people who are carrying this virus have a 200-fold increased risk of developing primary hepatocellular carcinoma. The typical pattern is that individuals infected with HBV in childhood develop chronic hepatitis, with the integration of part of the HBV genome into host cell chromosomes. Cirrhosis of the liver follows and eventually liver cancer develops some 20–50 years after the primary infection. The evidence suggests that the integration of viral DNA causes mutations in the host genes that regulate cell growth. This makes the liver cells more susceptible to exogenous carcinogens such as the fungal toxin aflatoxin and those generated by smoking tobacco. High alcohol consumption can be another co-factor. The other viruses that are strongly implicated in human cancers include hepatitis C virus with liver cancer; certain of the human papilloma viruses that cause warts on epithelial surfaces and cervical, penile and anal carcinomas; human T-cell lymphotrophic virus type 1 with adult T-cell leukaemia/lymphoma syndrome and HIV with Kaposi’s sarcoma.
11 The human immunodeficiency virus (HIV) HIV is an enveloped RNA-containing virus. It has a cone-shaped nucleocapsid containing two copies of a positive-sense single-stranded RNA molecule and the enzyme reverse transcriptase. Some 70 glyco-
Viruses
protein spikes project from the envelope. These contain the binding site for the CD4 protein present on the surface of T4-helper lymphocytes and ensure that the virus attaches to these cells. The main way in which people become infected is by the transmission of free virus or infected CD4+ T lymphocytes during heterosexual and male homosexual activity. Infection can also result from infected blood, particularly between drug abusers who share hypodermic needles. Health professionals whose work might bring them into contact with infected blood are also at risk. In the days before the screening of blood donors for HIV, clotting factor VIII prepared from infected blood also infected people with haemophilia. Vertical transmission also occurs, about 20% of babies born to HIV-positive mothers are infected. In the 20 years since the first reports, acquired immune deficiency syndrome (AIDS) has developed into one of the most devastating diseases to afflict the human population. The World Health Organization estimated that by December 2001 more than 60 million people had become infected with HIV and that over 20 million had died of the disease. The problem is critical in sub-Saharan Africa where 2.3 million people died of AIDS in 2001. After the primary infection, the virus is transported to lymph nodes where it replicates extensively in CD4+ lymphocytes. The number of these white cells declines and high titres of virus are found in the blood. Most patients experience a brief glandular fever-like illness at this stage, with characteristically persistent swollen lymph nodes. The number of the virus-specific CD8+ cytotoxic T lymphocytes then increases and this is associated with a reduction in the viral load in the blood to a low constant level; the population of CD4+ cells also recovers. There then follows an asymptomatic phase, which can last anything from 1 to 15 years. During this phase, most of the infected cells are located in lymph nodes where the virus is replicating actively. Enormous numbers of viral particles (1010) are produced per day, but the CD8+ cytotoxic lymphocytes destroy virus-infected cells and more CD4+ cells replace those killed. In this way the concentration of virus in the bloodstream is maintained at relatively low levels, although patients are infectious. As the years go by, it becomes increasing difficult to replace the CD4+ helper lymphocytes. It also seems
that HIV mutates, producing virions that become progressively more variable and difficult for the immune system to control. As a result, the number of virus particles in the blood increases and the population of CD4+ cells gradually declines. When the count of these cells falls below 200 per ml of blood, the immune system becomes seriously compromised and the individual becomes susceptible to infection by a range of opportunist pathogens including fungi, protozoa, bacteria and other viruses. Infections with Candida albicans, Pneumocystis carinii, Mycobacterium tuberculosis, cytomegalovirus and EBV are particularly common and will eventually kill the patient. While the CD4+ cells are the primary target, the virus also attacks muscle tissue and cells in the peripheral and central nervous system, resulting in the final stages of the disease, in major weight loss, myopathy, dementia and collapse of brain function. The replication cycle of HIV has been investigated in great detail in the hope that it will reveal ways of inhibiting the production of progeny viruses. The process starts when the glycoprotein spikes (gp120) in the virus envelope recognize and bind to the CD4+ receptors in the cytoplasmic membranes of the T4 helper lymphocytes. The fusion of the viral envelope and the cell membrane then leads to the entry of the HIV core into the cell. The core is uncoated, releasing the two RNA molecules and the reverse transcriptase into the cytoplasm. The RNA is copied by reverse transcriptase into a single strand of DNA, which is then duplicated to form a double-stranded DNA copy of the original viral RNA genome. This DNA moves into the host cell nucleus where it is integrated as proviruses into a host cell chromosome. These integrated proviruses can lie dormant in the cell or can be expressed, producing viral mRNA and proteins. The viral genome codes for envelope proteins (env), capsid proteins (gag) and enzymes involved in viral multiplication (pol). An important feature of HIV replication is that the polyprotein products of these genes have to be cleaved by a viral protease enzyme to produce the functional proteins. The viral proteins and new copies of the viral RNA are then assembled into new virions that bud off from the infected cell and can repeat the HIV replication cycle in other CD4+ cells. 79
Chapter 5
Enormous efforts have been made to develop chemotherapeutic agents for HIV. While there is no prospect of drugs that will eliminate the provirus from the populations of host lymphocytes, considerable progress has been made in the development of agents that preserve the stock of CD4+ cells in infected individuals and prevent the progression of the infection to AIDS. A major advance in this field was the discovery of the protease inhibitors such as saquinavir, ritonavir and indinavir. These compounds inhibit HIV replication by blocking the action of the protease that cleaves the viral polyproteins to produce the functional proteins. Currently a protease inhibitor is administered along with two reverse transcriptase inhibitors such as AZT, d4T or ddI. This triple therapy has been termed highly active anti-retroviral therapy (HAART). It reduces the numbers of HIV particles in the blood, restores lost immune functions and slows the progression to AIDS. Unfortunately, it has no effect on latently infected CD4+ so it does not clear the HIV infection. Patients who stop using the drugs experience a rapid rebound in levels of the virus in the blood and progression of the disease. The requirement for this life-long suppressive therapy makes the treatment extremely expensive. In the areas of the world where the disease is rampant, lack of funds makes the cost of treatment prohibitive. Despite huge investments of human and financial resources it has not yet proved possible to develop a vaccine that would protect against HIV infection. Research into HIV vaccines presents several particularly difficult problems. The imprecise operation of the viral reverse transcriptase enzyme ensures that antigenic variants are produced at high frequencies. The fact that HIV targets cells involved in the immune response brings special difficulties. For example, it might be thought that a virus-specific antibody that prevented the virus binding to its target host cell might produce a protective response. However, the binding of antibodies onto virus particles results in ingestion of the viruses by phagocytes and therefore infection of these cells. The current aim is to stimulate both virus-specific antibody and T-cell responses and to ensure that the vaccine produces the local mucosal immunity necessary to protect the genital and rectal epithelia. 80
Lack of an animal model and ethical considerations in the conduct of clinical trials have compounded the problem and slowed progress in vaccine development. The AIDS pandemic is out of control in parts of the world (sub-Saharan Africa and southern and south-east Asia) and the lack of access to effective chemotherapy means that the prognosis is bleak for the millions of people who are HIV-positive. Sexual intercourse is now the main mode of infection and if the pandemic is to be contained, sexually active individuals have to be persuaded to reduce the number of their partners and to practise safe sex using condoms.
12 Prions The causative agents of the neurodegenerative diseases bovine spongiform encephalopathy (BSE), scrapie in sheep and Creutzfeldt–Jakob disease (CJD) in humans used to be referred to as slow viruses. However, it is now clear that they are caused by a distinct class of infectious agents termed prions that have unique and disturbing properties. They can be recovered from the brains of infected individuals as rod-like structures which are oligomers of a 30-kDa glycoprotein. They are devoid of nucleic acid and are extremely resistant to heating and ultraviolet irradiation. They also fail to produce an immune response in the host. Just how such proteins can replicate and be infectious has only recently been understood. It seems that a glycoprotein (designated PrPc) with the same amino acid sequence as the prion (PrPsc) but with a different tertiary structure, is present in the membranes of normal neurons of the host. The evidence suggests that the prion form of the protein combines with the normal form and alters its configuration to that of the prion. The newly formed prion can then in turn modify the folding of other PrPc molecules. In this way the prion protein is capable of autocatalytic replication. As the prions slowly accumulate in the brain, the neurons progressively vacuolate. Holes eventually appear in the grey matter and the brain takes on a sponge-like appearance. The clinical symptoms take a long time to develop, up to 20 years in humans, but the disease
Viruses
has an inevitable progression to paralysis, dementia and death. It is now clear that the large-scale outbreak of BSE that began in the UK during the 1980s resulted from feeding cattle with supplements prepared from sheep and cattle offal. The recognition of this fact led to changes in animal feed policies and eventually to the imposition of a ban on the human consumption of bovine brain, spinal cord and lymphoid tissues that were considered to be potentially infectious. Unfortunately people had been consuming potentially contaminated meat for a number of years. Concerns that the agent had already been disseminated to humans in the food chain were realized in 1996 with the advent of a novel human disease that was called variant or vCJD. This condition was unusual as it attacked young adults with an average age of 30 rather than 60-year-olds that typically succumb to classical sporadic CJD. Studies on the experimental transmission of prions to mice provided evidence that vCJD represents infection by the BSE agent. The pathology in the mouse brain induced by the vCJD agent and the incubation time of the disease are different from that of classical CJD and very similar to that of BSE. Gel electrophoresis of the polypeptides from the brains of infected mice revealed that the different transmissible spongiform encephalitis agents have characteristic molecular signatures. These signatures are based on the lengths of protease-resistant fragments and the glycosylation patterns on the prion molecules. The patterns from vCJD agent were very different patterns from those of the classical CJD but remarkably similar to those formed by BSE. Since 1996 there has been a slow but gradual increase in the numbers of confirmed cases of vCJD. By March 2002 the number of deaths in the UK from vCJD had reached 109. As the average incuba-
tion time for vCJD is not yet known, it is difficult to estimate how many more cases will develop. The measures taken to protect the public will hopefully have prevented any further human infections but sadly there is no effective treatment available for those who have already contracted the disease.
13 Further reading Balfour, H. H. (1999) Antiviral drugs. N Engl J Med, 340, 1255–1268. Belay, E. (1999) Transmissible spongiform encephalopathies in humans. Annu Rev Microbiol, 53, 283–314. Collinge, J., Sidle, K. C., Meads, J., Ironside, J. & Hill, A. F. (1996) Molecular analysis of prion strain variation and the aetiology of ‘new variant’ CJD. Nature, 383, 685–690. Dalgleish, A. G. (1991) Viruses and cancer. Br Med Bull, 47, 21–46. Dimmock, N. J., Easton, A. J. & Leppard, K. N. (2001) Introduction to Modern Virology, 5th edn. Blackwell Science, Oxford. Hanke, T. (2001) Prospect for a prophylactic vaccine for HIV. Br Med Bull, 58, 205–218. Levy, J. A. (1998) HIV and the Pathogenesis of AIDS, 2nd edn. ASM Press, Herndon, VA. McCance, D. J. (1998) Human Tumour Viruses. ASM Press, Washington, DC. Maillard, J-Y. (2001) Virus susceptibility to biocides: an understanding. Rev Med Microbiol, 12, 63–74. Morrison, L. (2001) The global epidemiology of HIV/AIDS. Br Med Bull, 58, 7–18. Oxford, J. S., Coates, A. R. M., Sia, D. Y., Brown, K. & Asad, S. Potential target sites for antiviral inhibitors of human immunodeficiency virus (HIV). J Antimicrob Chemother, 23, S9–S27. Pattison, J. (1998) The emergence of bovine spongiform encephalopathy. Emerg Infect Dis, 4, 390–394. Prusiner, S. B. (1996) Molecular biology and pathogenesis of prion diseases Trends Biochem Sci, 21, 482–487. Weber, J. (2001) The pathogenesis of HIV-I infection. Br Med Bull, 58, 61–72.
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Chapter 6
Protozoa Tim Paget 1 Introduction 1.1 Parasitism 1.2 Habitats 1.3 Physiology of parasitic protozoa 2 Blood and tissue parasites 2.1 Malaria 2.1.1 Disease 2.1.2 Life cycle 2.2 Trypanosomatids 2.2.1 American trypanosomiasis (Chagas disease) 2.2.2 African trypanosomiasis (sleeping sickness) 2.2.3 Cutaneous and mucocutaneous leishmaniasis 2.2.4 Visceral leishmaniasis (kala-azar) 2.3 Toxoplasma gondii 3 Intestinal parasites 3.1 Giardia lamblia (syn. intestinalis, duodenalis) 3.2 Entamoeba histolytica 3.3 Cryptosporidium parvum
4 Trichomonas and free-living amoebas 4.1 Trichomonas vaginalis 4.2 Free-living opportunist amoebas 5 Host response to infection 5.1 Immune response 5.2 Immune pathology 5.3 Immune evasion 6 Control of protozoan parasites 6.1 Chemotherapy 6.1.1 Mechanisms of action and selective toxicity 6.1.2 Drug resistance 6.2 Other approaches to control 6.2.1 Biological control 6.2.2 Vaccination 7 Acknowledgement
1 Introduction
1.2 Habitats
1.1 Parasitism Parasitism is a specific type of interaction between two organisms that has many features in common with other infectious processes, but host–parasite interactions often operate over a longer timescale than those seen with other pathogens. This extended process results in significant host–parasite interaction at the cellular and organismal level. It is known, for example, that some parasites alter the behaviour of the host, while others, such as Giardia lamblia, induce biochemical change in the host cells at the site of infection (the duodenal epithelium). Most parasites have a life cycle that often involves several hosts; this means that survival and transmission between different hosts requires the parasite to exhibit more than one physiologically distinct form.
Parasites inhabit a wide range of habitats within their hosts. Some parasites will inhabit only one site throughout their life cycle, but many move to various sites within the body. Such movement may require the formation of motile cellular forms, and it will produce a significant change in the physiology and morphology of the parasite as a result of environmental change. Parasites moving from the gut to other tissues, for example, will encounter higher levels of oxygen, changes in pH and significant exposure to the host immune response. When life cycles involve more than one host organism these changes are greater. The reasons for such movement include evasion of host immune attack and to aid transmission. 1.3 Physiology of parasitic protozoa Parasitic protozoa, like their free-living counterparts, are single-celled eukaryotic organisms that 82
Protozoa
utilize flagella, cilia or amoeboid movement for motility. The complexity of some parasite life cycles means that some species may exhibit, at different times, more than one form of motility. All pathogenic protozoa are heterotrophs, using carbohydrates or amino acids as their major source of carbon and energy. Some parasitic protozoa utilize oxygen to generate energy through oxidative phosphorylation, but many protozoan parasites lack mitochondria, or have mitochondria that do not function like those in mammalian cells. As a result of this adaptation many parasites exhibit a fermentative metabolism that functions even in the presence of oxygen. The reason for the utilization of less efficient fermentative pathways is not clear, but it is presumably due in part to the fact that such parasites survive in environments where oxygen is only present occasionally or at low levels. For some parasites oxygen is toxic, and they appear to utilize it possibly in an effort to remove it and thus maintain an anaerobic metabolism. The metabolism of parasites is highly adapted, with many possessing unique organelles such as kienetoplasts and hydrogenosomes. Many synthetic pathways that are found in other eukaryotes are absent because many metabolic intermediates or precursors such as lipids, amino acids and nucleotides are actively scavenged from their environment. This minimizes energy expenditure, which is finely balanced in parasites and means that the membrane of parasitic protozoa is rich in transporters. Secretion of haemolysins, cytolysins, proteolytic enzymes, toxins, antigenic and immunomodulatory molecules that reduce host immune response also occurs in pathogenic protozoa. Survival of parasites is partly due to their high rate of reproduction, which may be either sexual or asexual; some organisms such as Plasmodium exhibit both forms of reproduction in their life cycle. Simple fission is characteristic of many amoeba, but some species also undergo nuclear division in the cystic state (cysts are forms required for survival outside the host) with each nucleus giving rise to new trophozoites (the growing, motile and pathogenic form).
2 Blood and tissue parasites This section considers the life cycles, disease and pathology of some blood and tissue parasites; this is not an exhaustive list but covers some of the most important species. These diseases are commonly associated with travel to tropical and subtropical countries, but diseases such as leishmaniasis are frequently seen in southern Spain and France. It should also be noted that climate change is altering the geographical distribution of many parasitic diseases. 2.1 Malaria Malaria has been a major disease of humankind for thousands of years. Despite the availability of drugs for treatment, malaria is still one of the most important infectious diseases of humans, with approximately 200–500 million new cases and 1–2.5 million deaths each year. Protozoa of the genus Plasmodium cause malaria and four species are responsible for the disease in humans: P. falciparum, P. vivax, P. ovale and P. malariae. P. falciparum and P. vivax account for the vast majority of cases, although P. falciparum causes the most severe disease. Other species of plasmodia infect reptiles, birds and other mammals. Malaria is spread to humans by the bite of female mosquitoes of the genus Anopheles but transmission by inoculation of infected blood and through congenital routes is also seen. These mosquitoes feed at night and their breeding sites are primarily in rural areas. 2.1.1 Disease The most common symptom of malaria is fever, although chills, headache, myalgia and nausea are frequently seen and other symptoms such as vomiting, diarrhoea, abdominal pain and cough occasionally appear. In all types of malaria, the periodic febrile response (fever) is caused by rupture of mature schizonts (one of the cell forms arising as part of the life cycle). In P. vivax and P. ovale malaria fever occurs every 48 hours, whereas in P. malariae, maturation occurs every 72 hours. In falciparum malaria fever may occur every 48 hours, but is usually irregular, showing no distinct periodicity. Apart 83
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from anaemia, most physical findings in malaria are often non-specific and offer little aid in diagnosis, although enlargement of some organs may be seen after prolonged infection. If the diagnosis of malaria is missed or delayed, especially with P. falciparum infection, potentially fatal complicated malaria may develop. The most frequent and serious complications of malaria are cerebral malaria and severe anaemia. 2.1.2 Life cycle Plasmodia have a complex life cycle (Fig. 6.1) involving a number of life cycle stages and two hosts. The human infective stage comprises the sporozoites (approximately 1 ¥ 7 mm), which are produced by sexual reproduction in the midgut of the anopheline mosquito (vector) and migrate to its salivary gland. When an infected Anopheles mosquito bites a human, sporozoites are injected into the bloodstream and are thought to enter liver parenchymal cells within 30 minutes of inoculation. In these cells the parasite differentiates into a spherical, multinucleate schizont which may contain 2000–40 000 uninucleate merozoites. This process of growth and development is termed exoerythrocytic schizogony. This exo-erythrocytic phase usually takes between 5 and 21 days, depending on the species of Plasmodium; however, in P. vivax and P. ovale the maturation of schizonts may be delayed for up to 1–2 years. These ‘quiescent’ parasites are called hypnozoites. Clinical illness is caused by the erythrocytic stage of the parasite life cycle; no disease is associated with sporozoites, the developing liver stage of the parasite, the merozoites released from the liver, or gametocytes. The common symptoms of malaria are due to the rupture of erythrocytes when erythrocytic schizonts mature (Fig. 6.2a). This release of parasite material triggers a host immune response, which in turn induces the formation of inflammatory cytokines, reactive oxygen intermediates and other cellular products. These pro-inflammatory molecules play a prominent role in pathogenesis, and are probably responsible for the fever, chills, sweats, weakness and other systemic symptoms associated with malaria. In P. falciparum malaria, infected erythrocytes adhere to the endothelium of capillaries 84
and postcapillary venules, leading to obstruction of the microcirculation and localized anoxia. The pathogenesis of anaemia appears to involve haemolysis or phagocytosis of parasitized erythrocytes and ineffective erythropoiesis. 2.2 Trypanosomatids The family Trypanosomatidae consists of two genera, Trypanosoma and Leishmania. These are important pathogens of humans and domestic animals and the diseases they cause constitute serious medical and economic problems. Because these protozoans have a requirement for haematin obtained from blood, they are called haemoflagellates. The life cycles of both genera involve insect and vertebrate hosts and have up to eight life cycle stages, which differ in the placement and origin of the flagellum. Trypanosomatids have a unique organelle called the kinetoplast. This appears to be a special part of the mitochondrion and is rich in DNA. Two types of DNA molecules, maxicircles that encode mainly certain important mitochondrial enzymes, and minicircles, which serve a function in the process of RNA editing, have been found in the kinetoplast. Replication of trypanosomatids occurs by single or multiple fission, involving first the kinetoplast, then the nucleus, and finally the cytoplasm. There are four major diseases associated with this group: Chagas disease is caused by Trypanosoma cruzi; sleeping sickness (African trypanosomiasis) is associated with Trypanosoma brucei; cutaneous and mucocutaneous leishmaniasis are caused by a range of species including Leishmania tropica, Leishmania major, Leishmania mexicana, Leishmania amazonensis and Leishmania braziliensis; and visceral leishmaniasis, which is also known as kala-azar, is typically caused by Leishmania donovani. 2.2.1 American trypanosomiasis (Chagas disease) Chagas disease begins as a localized infection that is followed by parasitaemia and colonization of internal organs and tissues. Infection may first be evidenced by a small tumour (chagoma) on the skin. Symptoms of the disease include fever, oedema and
Protozoa Human liver stages Liver cell
2
Infected liver cell
Mosquito stages 12
Ruptured oocyst
1
i
A
Mosquito takes a blood meal
Exo-erythrocytic cycle
(injects sporozoites) 11 Oocyst
Release of sporozoites
i
4
3
Ruptured schizont
Schizont
C Sporogonic cycle
Human blood stages Immature trophozoite (ring stage)
5 8
10 Ookinete
Mosquito takes a blood meal Macrogametocyte
d
(ingests gametocytes)
B Erythrocytic cycle Mature trophozoites
Macrogametocyte entering 9 macrogamete
d P. falciperum
Exflagellated macrogametocyte
Schizont d
7
Gametocytes d
7
Gametocytes i
= Infective stage
d
= Diagnostic stage
P. vivax P. ovale P. malariae
Fig. 6.1 The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infected female Anopheles mosquito
inoculates sporozoites into the human host (1). Sporozoites infect liver cells (2) and mature into schizonts (3), which rupture and release merozoites (4). (Of note, in P. vivax and P. ovale a dormant stage (hypnozoites) can persist in the liver and cause relapses by invading the bloodstream weeks, or even years later.) After this initial replication in the liver (exo-erythrocytic schizogony, A), the parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizogony, B). Merozoites infect red blood cells (5). The ring stage trophozoites mature into schizonts, which rupture releasing merozoites (6). Some parasites differentiate into sexual erythrocytic stages (gametocytes) (7). Blood stage parasites are responsible for the clinical manifestations of the disease. The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by an Anopheles mosquito during a blood meal (8). The parasite’s multiplication in the mosquito is known as the sporogonic cycle (C). While in the mosquito’s stomach, the microgametes penetrate the macrogametes, generating zygotes (9). The zygotes in turn become motile and elongated (ookinetes) (10), which invade the midgut wall of the mosquito where they develop into oocysts (11). The oocysts grow, rupture and release sporozoites (12), which make their way to the mosquito’s salivary glands. Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle (1).
myocarditis (infection of the heart muscle) with or without heart enlargement, and meningoencephalitis in children. The acute disease is frequently subclinical and patients may become asymptomatic carriers; this chronic phase may result, after 10–20 years, in cardiopathy. Chagas
disease is transmitted by cone-nosed triatomine bugs of several genera (Triatoma, Rhodnius and Panstrongylus) and in nature the disease exists among wild mammals and their associated triatomines. Human trypanosomiasis is seen in almost all countries of the Americas, including the 85
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(c)
(a) (b)
(e)
(g)
(d)
(f) Fig. 6.2 (a) Mature schizonts of Plasmodium vivax. P. vivax schizonts are large, have 12–24 merozoites, and may fill the red
blood cell (RBC). RBCs are enlarged 1.5–2 times and may be distorted. Under optimal conditions Schüffner’s dots may be seen. (b) Leishmania tropica amastigotes within an intact macrophage. (c) Toxoplasma gondii trophozoites in the bronchial secretions from an HIV-infected patient. (d) Trophozoites of Giardia intestinalis. Each cell has two nuclei and is 10–20 mm in length. (e) Trophozoites of Entamoeba histolytica with ingested erythrocytes, which appear as dark inclusions. (f) Oocysts of Cryptosporidium parvum (upper left) and cysts of Giardia intestinalis (lower right) labelled with immunofluorescent antibodies. (g) Trophozoites of Trichomonas vaginalis.
southern United States, but the main foci are in poor rural areas of Latin America. T. cruzi exhibits two cell types in vertebrate hosts, a blood form termed a trypomastigote, and in the tissues (mainly heart, skeletal and smooth muscle, and reticulo-endothelial cells) the parasite occurs as an amastigote (Fig. 6.3). Trypomastigotes 86
ingested when the insect takes a blood meal from an infected host transform into epimastigotes in the intestine. Active reproduction occurs and in 8–10 days, metacyclic trypomastigote forms appear which are flushed out of the gut with the faeces of the insect. These organisms are able to penetrate the vertebrate host only through the mucosa or
Protozoa Triatomine bug stages 1
Human stages
Triatomine bug takes a blood meal (passes metacyclic trypomastigotes in faeces; trypomastigotes enter bite wound or mucosal membranes such as the conjunctiva)
Metacyclic trypomastigote in hindgut
2 Metacyclic trypomastigotes
penetrate various cells at bite wound site. Inside cells they transform into amastigotes
i
8
7 Multiplies in midgut
5 6 Epimastigote stage
Triatomine bug takes a blood meal (trypomastigotes ingested)
Trypomastigotes can infect other cells and transform into intracellular amastigotes in new infection sites. Clinical manifestations can result from this infective cycle
3 Amastigotes multiply
by binary fission in cells of infected tissues
in midgut
d i
= Infective stage
d = Diagnostic stage
Intracellular amastigotes 4 transform into trypomastigotes, then burst out of the cell and enter the bloodstream
Fig. 6.3 An infected triatomine insect vector (or ‘kissing’ bug) takes a blood meal and releases trypomastigotes in its faeces near
the site of the bite wound. Trypomastigotes enter the host through the wound or through intact mucosal membranes, such as the conjunctiva (1). Common triatomine vector species for trypanosomiasis belong to the genera Triatoma, Rhodinius and Panstrongylus. Inside the host, the trypomastigotes invade cells, where they differentiate into intracellular amastigotes (2). The amastigotes multiply by binary fission (3) and differentiate into trypomastigotes, and then are released into the circulation as bloodstream trypomastigotes (4). Trypomastigotes infect cells from a variety of tissues and transform into intracellular amastigotes in new infection sites. Clinical manifestations can result from this infective cycle. The bloodstream trypomastigotes do not replicate (different from the African trypanosomes). Replication resumes only when the parasites enter another cell or are ingested by another vector. The ‘kissing’ bug becomes infected by feeding on human or animal blood that contains circulating parasites (5). The ingested trypomastigotes transform into epimastigotes in the vector’s midgut (6). The parasites multiply and differentiate in the midgut (7) and differentiate into infective metacyclic trypomastigotes in the hindgut (8). Trypanosoma cruzi can also be transmitted through blood transfusions, organ transplantation, transplacentally, and in laboratory accidents.
abrasions of the skin; hence, transmission does not necessarily occur at every blood meal. Within the vertebrate the trypomastigotes transform into amastigotes, which, after a period of intracellular multiplication at the portal of entry, are released into the blood as trypanosomes; these invade other cells or tissues, becoming amastigotes again.
The pathology of the infection is associated with inflammatory reactions in infected tissues. These can lead to acute myocarditis and destructionspecific ganglia. Parasite enzymes may also cause cell and tissue damage. In the absence of parasites, an autoimmune pathological process seems to be mediated by T lymphocytes (CD4+) (see Chapter 8) and by the production of certain cytokines; these in87
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duce a polyclonal activation of B lymphocytes and the secretion of large quantities of autoantibodies. 2.2.2 African trypanosomiasis (sleeping sickness) Sleeping sickness (African trypanosomiasis) is caused by Trypanosoma brucei, of which there are two morphologically indistinguishable subspecies: T. brucei rhodesiense and T. brucei gambiense. After infection the parasite undergoes a period of local multiplication then enters the general circulation via the lymphatics. Recurrent fever, headache, lymphadenopathy and splenomegaly may occur. Later, signs of meningo-encephalitis appear, followed by somnolence (sleeping sickness), coma and death. T. brucei, unlike T. cruzi, multiplies in the blood or cerebrospinal fluid. Trypanosomes ingested by a feeding fly must reach the salivary glands within a few days, where they reproduce actively as epimastigotes attached to the microvilli of the salivary gland until they transform into metacyclic trypomastigotes, which are found free in the lumen. Around 15–35 days after infection the fly becomes infective through its bite. The pathology of the infection is due to inflammatory changes associated with an induced autoimmune demyelination of nerve cells. Interestingly, the immunosuppressive action of components of the parasite’s membrane are probably responsible for frequent secondary infections such as pneumonia. Liberation of common surface antigens (the mechanism involved in immune evasion) in every trypanolytic crisis (episode of trypanosome lysis) leads to antibody and cell-mediated hypersensitivity reactions. It is believed that some cytotoxic and pathological processes are the result of biochemical and immune mechanisms.
2.2.3 Cutaneous and mucocutaneous leishmaniasis Leishmaniasis is the term used for diseases caused by species of the genus Leishmania that are transmitted by the bite of infected sandflies. The lesions of cutaneous and mucocutaneous leishmaniasis are 88
localized to the skin and mucous membranes. Visceral leishmaniasis is a much more severe disease, which involves the entire reticulo-endothelial system, and is discussed in section 2.2.4 of this chapter. Cutaneous leishmaniasis appears 2–3 weeks after the bite of an infected sandfly as a small cutaneous papule; this slowly develops and often becomes ulcerated and develops secondary infections. Secondary or diffuse lesions may develop. The disease is usually chronic but may occasionally be selflimiting. Leishmaniasis from a primary skin lesion may involve the oral and nasopharyngeal mucosa. Leishmania species that infect man are all morphologically similar and only exhibit one form, the intracellular amastigotes (3–6 mm long and 1.5–3 mm in diameter). Promastigotes (Fig. 6.2b) are found in the sandfly. In mammalian hosts amastigotes are phagocytosed by macrophages, but resist digestion and divide actively in the phagolysosome (Fig. 6.4). The female sandfly ingests parasites in the blood meal from an infected person or animal and these pass into the stomach where they transform into promastigotes, and multiply actively. The parasites attach to the walls of the oesophagus, midgut and hindgut of the fly, and some eventually reach the proboscis and are inoculated into a new host. The obvious symptoms of this infection are caused by the uptake of parasites by local macrophages. Host response to infection produces tubercle-like structures designed to limit the spread of infected cells. Some lesions may resolve spontaneously after a few months but other types of lesion may become chronic, sometimes with lymphatic and bloodstream dissemination. In infections due to L. braziliensis there is a highly destructive spread of infected macrophages to the oral or nasal mucosa. In L. mexicana, L. amazonensis and L. aethiopica infections the disease becomes more disseminated. The immunological response of the host plays an important factor in determining the precise pathology of the disease and this is apparent by the more severe type of infection seen in individuals with HIV. In Europe and Africa several rodents may act as reservoirs of the disease, but in countries such as India, transmission can occur in a man–sandfly–man cycle without rodent intervention. In rural semi-arid zones of Latin
Protozoa Triatomine bug stages
Human stages
1 Sandfly bug takes
a blood meal (injects promastigote stage into the skin) 8
2 Promastigotes
are phagocytosed by macrophages
Divide in midgut and migrate to proboscis
i 3
Promastigotes transform into amastigotes inside macrophages d 7 Amastigotes transform into
promastigote stage in midgut
4
Amastigotes multiply in cells (including macrophages) of various tissues d 6 Ingestion of
parasitized cells 5 Sandfly bug takes
i
= Infective stage
d = Diagnostic stage
a blood meal (ingests macrophages infected with amastigotes)
Fig. 6.4 Leishmaniasis is transmitted by the bite of female phlebotomine sandflies. The sandflies inject the infective stage,
promastigotes, during blood meals (1). Promastigotes that reach the puncture wound are phagocytosed by macrophages (2) and transform into amastigotes (3). Amastigotes multiply in infected cells and affect different tissues, depending in part on the Leishmania species (4). This originates the clinical manifestations of leishmaniasis. Sandflies become infected during blood meals on an infected host when they ingest macrophages infected with amastigotes (5, 6). In the sandfly’s midgut, the parasites differentiate into promastigotes (7), which multiply and migrate to the proboscis (8).
America, both wild and domestic dogs enter the epidemiological chain and the vector is a common sandfly, Lutzomyia longipalpis, abundant in and around houses. The disease is more common in children in both Latin America and the Mediterranean area. 2.2.4 Visceral leishmaniasis (kala-azar) Like cutaneous leishmaniasis, visceral leishmaniasis begins with a nodule at the site of inoculation but this lesion rarely ulcerates and usually disappears in a few weeks. However, symptoms and signs of systemic disease such as undulating fever, malaise,
diarrhoea, organ enlargement and anaemia subsequently develop. In more serious cases of visceral leishmaniasis the parasites, which can resist the internal body temperature, invade internal organs (liver, spleen, bone marrow and lymph nodes), where they occupy the reticulo-endothelial cells. The pathogenic mechanisms of the disease are not fully understood, but enlargement occurs in those organs that exhibit marked cellular alteration such as hyperplasia. Parasitized macrophages replace tissue in the bone marrow. Patients with advanced disease are prone to superinfection with other organisms.
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2.3 Toxoplasma gondii The term coccidia describes a group of protozoa that contains the organism Cryptosporidium (see intestinal parasites) as well as a number of important veterinary parasites. Toxoplasma gondii is an intestinal coccidian but the major pathology of infection is associated with other tissues and organs. T. gondii infects members of the cat family as definitive hosts and has a wide range of intermediate hosts. Infection is common in many warm-blooded animals, including humans. In most cases infection is asymptomatic, but devastating disease can occur congenitally in children as a result of infection during pregnancy. T. gondii infection in humans is a worldwide problem, although the rates of human infection vary from country to country. The reasons for these variations are thought to be environmental factors, cultural habits and the presence of domestic and native animal species. The frequency of postnatal toxoplasmosis acquired by eating raw meat and by ingesting food contaminated by oocysts from cat faeces (oocyst formation is greatest in the domestic cat) is unknown but it likely to be high. Widespread natural infection is possible because infected animals may excrete millions of resistant oocysts, which can survive in the environment for prolonged periods (months–years). Mature oocysts are approximately 12 mm in diameter and contain eight infective sporozoites. T. gondii infection in most animals including humans is asymptomatic. However, severe disease in humans is observed only in congenitally infected children and in immunosuppressed individuals. The most common symptom associated with postnatal infection in humans is lymphadenitis which may be accompanied by fever, malaise, fatigue, muscle pains, sore throat and headache (flu-like symptoms). Typically infection resolves spontaneously in weeks or months, but in immunosuppressed individuals, a fatal encephalitis may occur producing symptoms such as headache, disorientation, drowsiness, hemiparesis, reflex changes and convulsions. Prenatal T. gondii infections often target the brain and retina and can cause a wide spectrum of clinical disease. Mild disease may consist of impaired vision, whereas severely diseased children may exhibit a ‘classic tetrad’ of signs: 90
retinochoroiditis, hydrocephalus, convulsions and intracerebral calcifications. Hydrocephalus is the least common but most dramatic lesion of congenital toxoplasmosis. The life cycle of T. gondii was only fully described in the early 1970s when felines including domestic cats were identified as the definitive host. Various warm-blooded animals can serve as intermediate hosts. T. gondii is transmitted by three mechanisms: congenitally, through the consumption of uncooked infected meat and via faecal matter contamination. Figure 6.5 shows the life cycle of T. gondii. Cats acquire toxoplasma by ingesting any of three infectious stages of the organism: the rapidly multiplying forms, tachyzoites, the dormant bradyzoites (cysts) in infected tissue and the oocysts shed in faeces. The probability of infection and the time between infection and the shedding of oocysts varies with the stage of T. gondii ingested. Fewer than 50% of cats shed oocysts after ingesting tachyzoites or oocysts, whereas nearly all cats shed oocysts after ingesting bradyzoites. When a cat ingests tissue cysts, the cyst wall is dissolved by intestinal and gut proteolytic enzymes, which causes the release of bradyzoites. These enter the epithelial cells of the small intestine and initiate the formation of numerous asexual generations before the sexual cycle begins. At the same time that some bradyzoites invade the surface epithelia, other bradyzoites penetrate the lamina propria and begin to multiply as tachyzoites (trophozoites) (Fig. 6.2c). Within a few hours, tachyzoites may disseminate to other tissues through the lymph and blood. Tachyzoites can enter almost any type of host cell and multiply until it is filled with parasites; the cell then lyses and releases more tachyzoites to enter new host cells. The host usually controls this phase of infection, and as a result the parasite enters the ‘resting’ stage in which bradyzoites are isolated in tissue cysts. Tissue cysts are formed most commonly in the brain, liver and muscles. These cysts usually cause no host reaction and may remain dormant for the life of the host. In intermediate hosts, such as humans, the extra-intestinal cycle of T. gondii is similar to the cycle in cats except that there is no sexual stage. Most cases of toxoplasmosis in humans are probably acquired by the ingestion of either tissue cysts in infected meat or oocysts in food contaminated
Protozoa
1
i Faecal
i Tissue
oocysts
cysts
Both oocysts and tissue cysts transform into tachyzoites shortly after ingestion. Tachyzoites localize in neural and muscle tissue and develop into tissue cyst bradyzoites. If a pregnant woman becomes infected, tachyzoites can infect the fetus via the bloodstream 3
i
2
= Infective stage
d = Diagnostic stage d
d Serum, CSF
d Diagnostic stage 1) Serological diagnosis or 2) Direct identification of the parasite from peripheral blood, amniotic fluid, or in tissue sections
Fig. 6.5 Members of the cat family (Felidae) are the only known definitive hosts for the sexual stages of Toxoplasma gondii and thus are the main reservoirs of infection. Cats become infected with T. gondii by carnivorism (1). After tissue cysts or oocysts are ingested by the cat, viable organisms are released and invade epithelial cells of the small intestine where they undergo an asexual cycle followed by a sexual cycle and then form oocysts, which are then excreted. The unsporulated oocyst takes 1–5 days after excretion to sporulate (become infective). Although cats shed oocysts for only 1–2 weeks, large numbers may be shed. Oocysts can survive in the environment for several months and are remarkably resistant to disinfectants, freezing and drying, but are killed by heating to 70°C for 10 minutes. Human infection may be acquired in several ways: (A) ingestion of undercooked infected meat containing Toxoplasma cysts (2); (B) ingestion of the oocyst from faecally contaminated hands or food (3); (C) organ transplantation or blood transfusion; (D) transplacental transmission; (E) accidental inoculation of tachyzoites. The parasites form tissue cysts, most commonly in skeletal muscle, myocardium and brain; these cysts may remain throughout the life of the host.
with cat faeces. Bradyzoites from the tissue cysts or sporozoites released from oocysts invade intestinal epithelia and multiply. T. gondii may spread both locally to mesenteric lymph nodes and to distant organs by invading the lymphatic and blood systems. Focal areas of necrosis (caused by localized cell
lysis) may develop in many organs. The extent of the disease is usually determined by the extent of injury to infected organs, especially to vital and vulnerable organs such as the eye, heart and adrenals. Opportunist toxoplasmosis in immune suppressed patients usually represents reactivation of 91
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chronic infection. The predominant lesion of toxoplasmosis — encephalitis in these patients — is necrosis, which often results in multiple abscesses, some as large as a tennis ball.
3 Intestinal parasites Gut protozoan parasites include Entamoeba histolytica, Giardia lamblia, Dientamoeba fragilis, Balantidium sp., Isospora sp. and Cryptosporidium parvum. All these organisms are transmitted by the faecal–oral route and most of them are cosmopolitan in their distribution. A good example of this is Giardia, which is found in nearly all countries of the world. In many developed countries, including the UK and USA, it is one of the most commonly identified waterborne infectious organisms. Cryptosporidium, like Toxoplasma, has a complex life cycle utilizing both sexual and asexual reproduction. In contrast, Giardia and Entamoeba have simple life cycles utilizing only asexual reproduction. These latter organisms are members of a small group of eukaryotes that do not have mitochondria. It had long been assumed that they never had mitochondria, but recent studies showing the presence of mitochondrial-like enzymes and structural proteins suggest that it is more likely that this organelle was lost as a result of metabolic/physiological adaptation. 3.1 Giardia lamblia (syn. intestinalis, duodenalis) Giardia lamblia is the causative agent of giardiasis — a severe diarrhoeal disease. The incidence of Giardia infection worldwide ranges from 1.5% to 20% but is probably significantly higher in countries where standards of hygiene are poor. The most common route of spread is via the faecal–oral route, although spread can also occur through ingestion of contaminated water and these modes of transmission are particularly prevalent in institutions, nurseries and day-care centres. Recent outbreaks and epidemics in the UK, USA and Eastern Europe have been caused by drinking contaminated water from community water supplies or directly from rivers and streams. Many animals harbour 92
Giardia species that are indistinguishable from G. lamblia, and this has raised the question of the existence of animal reservoirs of Giardia. Recent findings of Giardia-infected animals in watersheds from which humans acquired giardiasis, and the successful interspecies transfer of these organisms suggests that giardiasis is a zoonotic infection. More recently, it has been recognized that Giardia infection may be transmitted by sexual activity, particularly among homosexual men. This organism exhibits only two life cycle forms: the vegetative bi-nucleate trophozoite (10–20 mm long by 2–3 mm wide) (Fig. 6.2d) and the transmissible quadra-nucleate cyst (10–12 mm long by 1–3 mm wide). Trophozoites have four pairs of flagella and an adhesive disc, which is thought to help adhesion to the intestinal epithelium. Division in trophozoites is by longitudinal fission. It was long believed that Giardia was a nonpathogenic commensal. However, we now know that Giardia can produce disease ranging from a self-limiting diarrhoea to a severe chronic syndrome. Immune-competent individuals with giardiasis may exhibit some or all of the following signs and symptoms: diarrhoea or loose, foul-smelling stools; steatorrhoea (fatty diarrhoea); malaise; abdominal cramps; excessive flatulence; fatigue and weight loss. Infected individuals with an immune deficiency or protein-calorie malnutrition may develop a more severe disease and will exhibit symptoms such as interference with the absorption of fat and fat-soluble vitamins, retarded growth, weight loss, or a coeliac disease-like syndrome. Giardia infection is initiated by ingestion of viable cysts (Fig. 6.6); the infective dose of which can be a low as one cyst, although infection initiated by 10–100 viable cysts is more likely. As the cysts pass through the stomach the low pH and elevated CO2 induce excystation (cyst–trophozoite transformation). From each cyst two complete trophozoites emerge and these rapidly undergo division then attach to the duodenal and jejunal epithelium. Once attached, they will undergo division, and 4–7 days later they will detach and begin to round up and form cysts (encystment). This process is thought to be induced in response to bile. The first cysts are found in faeces after 7–10 days. The underlying pathology of giardiasis is not
Protozoa
2
i
Fig. 6.6 Cysts are resistant forms and
are responsible for transmission of giardiasis. Both cysts and trophozoites can be found in the faeces (diagnostic stages) (1). The cysts are hardy, and can survive several months in cold water. Infection occurs by the ingestion of cysts in contaminated water, food or by the faecal–oral route (hands or fomites) (2). In the small intestine, excystation releases trophozoites (each cyst produces two trophozoites) (3). Trophozoites multiply by longitudinal binary fission, remaining in the lumen of the proximal small bowel where they can be free or attached to the mucosa by a ventral sucking disc (4). Encystation occurs as the parasites transit toward the colon. The cyst is the stage found most commonly in non-diarrhoeal faeces (5). Because the cysts are infectious when passed in the stool or shortly afterwards, person-to-person transmission is possible. While animals are infected with Giardia, their importance as a reservoir is unclear.
Contamination of water, food, or hands/fomites with infective cysts
Trophozoites are also passed in stool but they do not survive in the environment
1
i
= Infective stage
d = Diagnostic stage
i d
d Cyst
3
Cyst
4
5
Trophozoites
fully understood. The trophozoites do not invade the mucosa, and although their presence may have some physical effects on the surface it is more likely that some of the pathology is caused by inflammation of the mucosal cells of the small intestine causing an increased turnover rate of intestinal mucosal epithelium. The immature replacement cells have less functional surface area and less digestive and absorptive ability. This would account for the microscopic changes seen in infected epithelia. It has been suggested that other mechanisms may exist, e.g. toxin production, but to date no such molecule has been observed. 3.2 Entamoeba histolytica Entamoeba histolytica is the causative agent of
amoebic dysentery; another infection transmitted via the faecal–oral route. The severity of this and related pathologies caused by this organism can vary from diarrhoea associated with the intestinal infection to extra-intestinal amoebiasis producing hepatic and often lung infection. The prevalence of amoebiasis in developing countries reflects the lack of adequate sanitary systems. It had long been known that most infections associated with E. histolytica are asymptomatic, but in the late 1980s a separate, but morphologically and biochemically similar species, E. dispar, was identified. This was shown to be non-pathogenic but commonly misidentified as E. histolytica and thus the cause of ‘asymptomatic Entamoeba infection’. E. histolytica has a relatively simple life cycle and, like Giardia, exhibits only two morphological 93
Chapter 6
forms: the trophozoite and cyst stages. Trophozoites (Fig. 6.2e) vary in size from 10 to 60 mm and are actively motile. The cyst is spherical, 10– 20 mm in diameter, with a thin transparent wall. Fully mature cysts contain four nuclei. Symptoms of amoebic dysentery are associated with mucosal invasion and ulceration. Mucosal erosion causes diarrhoea, the severity of which increases with the level of invasion and colonization. Symptoms can also be affected by the site of the infection. Peritonitis as a result of perforation has been reported in connection with severe amoebic infection. Extra-intestinal amoebiasis is usually associated with liver infection, causing abscesses and/or enlargement. The abscess appears as a slowly enlarging liver mass and will cause noticeable pain. Jaundice may also occur due to blockage of the bile. Pleural, pulmonary, and pericardial infection results from metastatic spread from the liver, but can also manifest in other parts of the viscera or give rise to a brain abscess. However, these complications are uncommon. The life cycle of E. histolytica (Fig. 6.7) is simple, but the ability of trophozoites to infect sites other than the intestine make it more complex than that of Giardia. Infection is initiated by ingestion of mature cysts, and again, excystation occurs during transit through the gut. After this, trophozoites rapidly divide by simple fission to produce four amoebic cells, which undergo a second division; thus each cyst yields eight trophozoites. Survival outside the host depends on the resistant cyst form. The pathology of the disease is only partially understood. The process of tissue invasion has been well studied and involves binding and killing of the host cells by specific adhesin molecules and the action of a pore-forming protein, amoebapore. The initial superficial ulcer may deepen into the submucosa and become chronic. Spread may occur by direct extension, by undermining of the surrounding mucosa until it sloughs, or by penetration that can lead to perforation. If the trophozoites gain access to the vascular or lymphatic circulation, metastases may occur first to the liver and then by direct extension or further metastasis to other organs, including the brain.
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3.3 Cryptosporidium parvum Cryptosporidium parvum is a ubiquitous coccidian parasite that causes cryptosporidiosis in man; however, other species are known to cause infection in immunocompromised patients. The life cycle of the parasite is complex but is completed in a single host. Infection follows the ingestion of oocysts associated with contaminated water or food. According to the World Health Organization (WHO) the health significance of C. parvum is high due to the persistence of the organism in the environment. Cattle represent the most important reservoir of C. parvum but other mammals, domestic and wild, can be infected and act as carriers of the disease, even if asymptomatic. It is now known that a number of genetically distinct subspecies exist that can be divided into two groups. Group I comprises organisms that are human infective only (soon to be reclassified as C. hominis). Group II comprises organisms that infect a wider range of hosts. There have been several large outbreaks of this infection in the UK and USA, in which water was identified as the initial vehicle for transmission. Members of this genus are intracellular parasites infecting the intestinal mucosal epithelium. The two major life cycle forms are the oval oocyst and the sporozoite. C. parvum infections are often asymptomatic, but symptoms such as profuse watery diarrhoea, stomach cramps, nausea, vomiting and fever are typical. The symptoms can last from several days to a few weeks in immunocompetent individuals, but in immunocompromised patients infection can become chronic, lasting months or even years. The mean infective dose for immunocompetent people is dependent on the strain of C. parvum but it is considered to be approximately 100 cells, and infants are more vulnerable to infection. Diarrhoea is a major cause of childhood mortality and morbidity as well as malnutrition in developing countries. Cryptosporidium is the third most common cause of infective diarrhoea in children in such countries, and consequently it plays a role in the incidence of childhood malnutrition. Cryptosporidium infection has a higher nutritional impact in boys than girls, due to an increased need for micronutrients in boys to build up larger muscle mass. However, breast-feeding does offer
Protozoa
i
= Infective stage 2
d = Diagnostic stage
i
d
A d
C d B d
1
Passed in faeces
i
d
A = Noninvasive colonization B = Intestinal disease C = Extraintestinal disease 4 Trophozoites
d
Exits host Excystation 3
Trophozoites 4
d 5 Cysts
d
d
d
d
Fig. 6.7 Cysts are passed in faeces (1). Infection by Entamoeba histolytica occurs by ingestion of mature cysts (2) in faecally
contaminated food, water or hands. Excystation (3) occurs in the small intestine and trophozoites (4) are released, which migrate to the large intestine. The trophozoites multiply by binary fission and produce cysts (5), which are passed in the faeces (1). Because of the protection conferred by their walls, the cysts can survive days to weeks in the external environment and are responsible for transmission. (Trophozoites can also be passed in diarrhoeal stools, but are rapidly destroyed once outside the body, and if ingested would not survive exposure to the gastric environment.) In many cases, the trophozoites remain confined to the intestinal lumen (A, non-invasive infection) of individuals who are asymptomatic carriers, passing cysts in their stool. In some patients the trophozoites invade the intestinal mucosa (B, intestinal disease), or through the bloodstream, extra-intestinal sites such as the liver, brain and lungs (C, extra-intestinal disease), with resultant pathological manifestations. It has been established that the invasive and non-invasive forms represent two separate species, respectively, E. histolytica and E. dispar. However, not all persons infected with E. histolytica will have invasive disease. These two species are morphologically indistinguishable. Transmission can also occur through faecal exposure during sexual contact (in which case not only cysts, but also trophozoites could prove infective).
some protection against infection. In immunocompromised individuals Cryptosporidium infection causes a severe gastroenteritis, and often the parasites infect other epithelial tissues causing pneumo-
nia; the mortality rate due to C. parvum in AIDS patients is between 50% and 70%. Infection occurs when oocysts (Fig. 6.2f) excyst following environmental stimuli (typical intestinal conditions) and 95
Chapter 6
parasitize the epithelial cells which line the intestine wall (Fig. 6.8). After several further stages of the cycle, two forms of oocyst are produced; soft-walled oocysts re-initiate infection of neighbouring enterocytes while hard-walled cysts are expelled in the faeces. Little is known about the mechanism by which these organisms cause disease. They are known to invade cells but this process is atypical in that the parasites form a vacuole just below the epithelial cell membrane. During infection a variety of changes are seen such as partial villous atrophy, crypt lengthening and inflammation; these responses are probably due in part to cell damage that occurs during the growth of the intracellular forms. It has also been proposed that parasite enzymes and/or immune-mediated mechanisms may also be involved. It should be remembered, however, that cryptosporidiosis is resolved by the immune system in healthy patients normally within 3 weeks.
4 Trichomonas and free-living amoebas 4.1 Trichomonas vaginalis Trichomonas vaginalis is a common sexually transmitted parasite. Infection rates vary from 10% to 50% with the highest reported rates found in the USA. Infections are usually asymptomatic or mild although symptomatic infection is most common in women. Trichomonads are all anaerobes and contain hydrogenosomes. This organelle is found in very few other anaerobic eukaryotes and is often
termed the ‘anerobic mitochondrion’. A number of functions have been assigned to it, and it has been shown to function in the generation of ATP. This organism does not exhibit a life cycle as only the motile (flagellate/amoeboid) trophozoite (Fig. 6.2g) has been seen and division is by binary fission. Trichomonads have a pear-shaped body 7–15 mm long, a single nucleus, three to five forward-directed flagella, and a single posterior flagellum that forms the outer border of an undulating membrane. Trichomoniasis in women is frequently chronic and is characterized by vaginal discharge and dysuria. The inflammation of the vagina is usually diffuse and is characterized by reddening of the vaginal wall and migration of polymorphonuclear leucocytes into the vaginal lumen (these form part of the vaginal discharge). Because there is no resistant cyst, transmission from host to host must be direct. The inflammatory response in trichomoniasis is the major pathology associated with this organism; however, the mechanisms of induction are not known. It is likely that mechanical irritation resulting from contact between the parasite and vaginal epithelium is a major cause of this response but the organism produces high concentrations of acidic end-products and polyamines, both of which would also irritate local tissues.
4.2 Free-living opportunist amoebas The free-living opportunist amoebas are an often forgotten group of protozoans. The two major
Fig. 6.8 Life cycle of Cryptosporidium. Sporulated oocysts, containing four sporozoites, are excreted by the infected host
䉴
through faeces and possibly other routes such as respiratory secretions (1). Transmission of Cryptosporidium parvum occurs mainly through contact with contaminated water (e.g. drinking or recreational water). Occasionally food sources, such as chicken salad, may serve as vehicles for transmission. Many outbreaks in the USA have occurred in water parks, community swimming pools and day-care centres. Zoonotic transmission of C. parvum occurs through exposure to infected animals or exposure to water contaminated by faeces of infected animals (2). Following ingestion (and possibly inhalation) by a suitable host (3), excystation (a) occurs. The sporozoites are released and parasitize epithelial cells (b, c) of the gastrointestinal tract or other tissues such as the respiratory tract. In these cells, the parasites undergo asexual multiplication (schizogony or merogony) (d, e, f) and then sexual multiplication (gametogony) producing microgamonts (male, g) and macrogamonts (female, h). Upon fertilization of the macrogamonts by the microgametes (i), oocysts (j, k) develop that sporulate in the infected host. Two different types of oocysts are produced, the thick-walled oocyst, which is commonly excreted from the host (j) and the thinwalled oocyst (k), which is primarily involved in autoinfection. Oocysts are infective upon excretion, thus permitting direct and immediate faecal–oral transmission. (From: Juranek DD. Cryptosporidiosis. In: Hunter’s Tropical Medicine, 8th edn. Edited by GT Strickland.)
96
Protozoa 3 Thick-walled oocyst ingested by host
Recreational water 2
Drinking water
Contamination of water and food with oocysts
1
j Thick-walled oocyst (sporulated) exits host
b Sporozoite
a Oocyst
Thick-walled oocyst (sporulated) exits host
c e
d
Auto-infection k Thin-walled oocyst (sporulated)
Type I Meront
Trophozoite
Asexual cycle
Merozoite Macrogametes
Microgametes f
Undifferentiated gamont
g
Type II Merozoite Macrogamont
Merozoites Sexual cycle
Zygote i
h
97
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groups, Naegleria and Acanthamoeba, infect humans and both can cause fatal encephalitis. Both types of infections are rare, with less than 200 cases of Naegleria fowleri infection recorded worldwide and approximately 100–200 cases of Acanthamoeba ulcerative keratitis per year. This disease is commonly associated with contact lens use and it is thought that infection is caused by a combination of corneal trauma and dirty contact lenses. Both types of amoeba produce resistant cysts and Naegleria also exhibits a flagellate form. Both Acanthamoeba and Naegleria are free-living inhabitants of fresh water and soil, but Naegleria fowleri (the human pathogen) reproduces faster in warm waters up to 46°C. Treatment of water by chlorination or ozonolysis does not entirely eliminate cysts and both amoebae have been isolated from air-conditioning units. Naegleria fowleri is the causative agent of primary amoebic meningo-encephalitis, a rapidly fatal disease that usually affects children and young adults. In all cases, contact with amoebae occurs as a result of swimming in infected fresh water. The organisms enter the brain via the olfactory tract after amoebae are inhaled or splashed into the olfactory epithelium. The incubation period ranges from 2 to 15 days and depends both on the size of the inoculum and the virulence of the strain. The disease appears with the sudden onset of severe frontal headache, fever, nausea, vomiting and stiff neck. Symptoms develop rapidly to lethargy, confusion and coma and in all cases to date the patient died within 48–72 hours. Acanthamoeba castellanii, A. culbertsoni and other pathogenic Acanthamoeba species can cause opportunist lung and skin infections in immunocompromised individuals. Where amoebae spread from such lesions to the brain, they can cause a slowly progressive and usually fatal encephalitis. In addition, Acanthamoeba can cause an ulcerating keratitis in healthy individuals, usually in association with improperly sterilized contact lenses. The presence of cysts and trophozoites in alveoli or in multiple nodules or ulcerations of the skin characterizes acanthamoebic pneumonitis and dermatitis. Spread of amoebae to the brain produces an encephalitis, characterized by neurological changes, drowsiness, personality changes and seizures in the 98
early stages of infection, which progress to altered mental status, lethargy and cerebellar ataxia, finally ending in coma and death. Acanthamoeba keratitis is characterized by painful corneal ulcerations that fail to respond to the usual anti-infective treatments. The infected and damaged corneal tissue may show a characteristic annular infiltrate and congested conjunctivae. If not successfully treated, the disease progresses to corneal perforation and loss of the eye or to a vascularized scar over thinned cornea, with impaired vision.
5 Host response to infection Mechanisms to control parasitic protozoa are similar to those utilized for other infectious agents; they can be divided into non-specific mechanism(s) and specific mechanism(s) involving the immune system. The best studied non-specific mechanisms include those that affect the entry of parasites into the red blood cell. The sickle cell haemoglobin trait and lack of the Duffy factor on the erythrocyte surface make the red cell more resistant to invasion by Plasmodium. These traits are commonly found in populations from malaria-endemic regions. A second example of a non-specific factor is the presence of trypanolytic factors in the serum of humans which confer resistance to T. brucei, Although nonspecific factors can play a key role in resistance, usually they work in conjunction with the host’s immune system. 5.1 Immune response Unlike most other types of infection, protozoan diseases are often chronic, lasting for months to years. When associated with a strong host immune response, this type of long-term infection is apt to result in a high incidence of immunopathology. Until recently the importance of host immune response in controlling many parasite infections was not fully appreciated, but the impact of HIV infection on many parasitic diseases has highlighted this relationship. Different parasites elicit different humoral and/or cellular immune responses. In malaria and trypanosome infections, antibody appears to play a
Protozoa
major role in immunity, although it would seem that for many organisms both humoral and cellular immunity are required for killing of parasites. Cellular immunity is believed to be the most important mechanism in the killing of Leishmania and Toxoplasma. Cytokines are involved in the control of both the immune response and also the pathology of many parasitic diseases. Helper (h) and cytotoxic (c) T cells play major roles in the induction/control of the response. The various subsets of these produce different profiles of cytokines. For example, the Th1 subset produces gamma interferon (IFN-g) and interleukin-2 (IL-2) and is involved in cell-mediated immunity. In contrast, the Th2 subset produces IL-4 and IL-6, and is responsible for antibody-mediated immunity. The induction of the correct T-cell response is key to recovery. The Th1 subset and increased IFN-g are important for the control of Leishmania, T. cruzi and Toxoplasma infections, whereas the Th2 response is more important in parasitic infections in which antibody is a major factor. It is important to recognize that the cytokines produced by one T-cell subset can up- or down-regulate the response of other T-cell subsets; IL-4 will down-regulate Th1 cells for example. The cytokines produced by T and other cell types do not act directly on the parasites but induce changes in the metabolism of glucose, fatty acid and protein in other host cells. Cytokines can also stimulate cell division and, therefore, clonal expansion of T- and B-cell subsets. This can lead to increased antibody production and/or cytotoxic T-cell numbers. The list of cytokines and their functions is growing rapidly, and it would appear that these chemical messages influence all phases of the immune response. They are also clearly involved in the multitude of physiological responses (fever, decreased food intake, etc.) observed in an animal’s response to a pathogen, and in the pathology that results. 5.2 Immune pathology The protozoa can elicit humoral responses in which antigen–antibody complexes are formed and these can trigger coagulation and complement systems. Immune complexes have been found circulating in serum and deposited in the kidneys where they may contribute to conditions such as glomerulonephri-
tis. In other tissues these complexes can also induce localized hypersensitivities. It is thought that this type of immediate hypersensitivity is responsible for various clinical syndromes including blood hyperviscosity, oedema and hypotension. Another important form of antibody-mediated pathology is autoimmunity. Autoantibodies to a number of different host antigens (e.g. red blood cells, laminin, collagen and DNA) have been demonstrated. These autoantibodies may play a role in the pathology of parasitic diseases by exerting a direct cytotoxic effect on the host cells, e.g. autoantibodies that coat red blood cells produce haemolytic anaemia; they may also cause damage through a build up of antigen–antibody complexes. Many parasites can elicit the symptoms of disease through the action of their surface molecules such as the pore-forming proteins of E. histolytica that induce contact-dependent cell lysis, and trypanosome glycoproteins that can fix and activate complement resulting in the production of biologically active and toxic complement fragments. A range of parasite-derived enzymes such as proteases and phospholipases can cause cell destruction, inflammatory responses and gross tissue pathology. 5.3 Immune evasion Parasites exhibit a number of mechanisms that allow them to evade host immune response. Two such mechanisms are displayed by trypanosomes, which are able to exhibit both antigenic masking and antigenic variation. Masking means the parasite becomes coated with host components and therefore is not recognized as foreign, and antigenic variation results in surface antigens being changed during the course of an infection, so again the immune response is evaded. Parasites can also suppress the host’s immune response either to the parasite specifically or to foreign antigens in general. This, however, can cause a number of problems, as general immune suppression may make the individual more susceptible to secondary infection.
6 Control of protozoan parasites It is now clear that the best approach for the suc99
Chapter 6 Table 6.1 Common anti-protozoal drugs and their modes of action Drug
Mode of action (if known)
Mechanism of selectivity
Target organism(s)
Dapsone Proguanil Pyrimethamine Sulphonamides Benznidazole Chloroquine Mefloquine Metronidazole Pentamidine Quinine Eflornithine Tetracycline Benzimadazoles Amphotericin B Atovaquone Melarasaprol
Co-factor synthesis Co-factor synthesis Co-factor synthesis Co-factor synthesis Nucleic acid synthesis Nucleic acid synthesis Nucleic acid synthesis Nucleic acid synthesis Nucleic acid synthesis Nucleic acid synthesis Protein function Protein function Microtubule function Membrane function Energy metabolism Energy metabolism
Plasmodium spp. Plasmodium spp. Plasmodium spp. Plasmodium spp. Trypanosoma spp. Plasmodium spp. Plasmodium spp. Giardia, Trichomonas, Entamoeba Leishmania spp. Plasmodium spp. T. brucei gambiense Plasmodium spp. Giardia, Trichomonas Leishmania spp. Plasmodium spp. T. brucei gambiense
Primaquine
Energy metabolism
Unique target Differences in the target Differences in the target Differences in the target Activation in the parasite Differential uptake Differential uptake Activation in the parasite Differential uptake Differential uptake Differences in the target Differential uptake Differences in the target Differences in the target Differences in the target Target pathway more important in the parasite Differences in the target
cessful control of parasites requires the integration of a number of methods, which draw upon our increasing understanding of the parasites’ life cycle, epidemiology and host response to infection. 6.1 Chemotherapy The origins of chemotherapy are closely linked to the development of anti-parasitic agents, but there has been slow progress in the development of new and novel anti-protozoal agents over the past 30 years. Recently, with the support of the WHO and government-sponsored research, new anti-parasitic drugs are slowly coming into the market. Interestingly there are still a number of protozoan parasite infections such as cryptosporidiosis for which there is no effective treatment. 6.1.1 Mechanisms of action and selective toxicity For many of the commonly used anti-protozoal drugs the modes of action and mechanisms of selective toxicity are well understood, although for some the precise mechanism remains unclear. The most 100
Trypanosoma spp.
common anti-protozoal drugs and their modes of action are shown in Table 6.1. Considering the drugs in relation to modes of action, dapsone and the sulphonamides block the biosynthesis of tetrahydrofolate by inhibiting dihydropteroate synthetase, while the 2,4-diaminopyrimidines (proguanil and pyrimethamine) block the same pathway but at a later step catalysed by dihydrofolate reductase. The drugs that interfere with nucleic acid synthesis include those that bind to the DNA and intercalate with it such as chloroquine, mefloquine and quinine, and pentamidine that is unable to intercalate but probably interacts ionically. Other compounds such as benznidazole and metronidazole may alkylate DNA through activation of nitro groups via a one electron reduction step. Several of these compounds, however, including chloroquine, mefloquine, quinine and metronidazole have more than one potential mode of action. Chloroquine, for example, inhibits the enzyme haem polymerase, which functions to detoxify the cytotoxic molecule haem that is generated during the degradation of haemoglobin. Metronidazole is reduced in the parasite cell and forms a number of cytotoxic interme-
Protozoa
diates, which can cause damage not only to DNA but also to membranes and proteins. Tetracycline targets protein synthesis in Plasmodium via a similar mechanism to that seen in bacteria: inhibition of chain elongation and peptide bond formation. Eflornithine interferes with the metabolism of the amino acid ornithine in T. brucei gambiense by acting as a suicide substrate for the enzyme ornithine decarboxylase. Albendazole has recently been shown to have significant anti-giardial activity, although its mode of action is unclear. In leishmania, amphotericin B binds to the membrane sterol ergosterol making the plasma membrane leaky to ions and small molecules (e.g. amino acids), while the anti-protozoal drugs atovaquone and primaquine bind to the cytochrome bc1 complex and inhibit electron flow. The anti-trypanosomal drug melarsaprol is most likely to act by blocking glycolytic kinases, especially the cytoplasmic pyruvate kinase, although they may also disrupt the reduction of trypanothione.
lation of pesticide residues in the food chain and pesticide resistance in the target organism. Window screens and bed nets do prevent mosquito bites, however, and there has been a lot of interest in using bed nets impregnated with insecticide. Environmental control was a major form of control used before the development of modern insecticides. A good example of this is mosquito control through the removal of breeding sites by drainage, land reclamation projects, removal of vegetation overhanging water, speeding up water flow in canals and periodic drainage and drying out of canals. Life cycle forms that enter the water system, such as cysts and oocysts of Giardia and Cryptosporidium, can present a major public health problem. These forms are often resistant to common disinfection methods and require physical removal from waters. Cysts and oocysts can be destroyed by use of proper sewage treatments like anaerobic digestion, but these systems require regular maintenance in order to remain effective. 6.2.1 Biological control
6.1.2 Drug resistance As with bacteria, drug resistance in some parasites such as Plasmodium is a major problem and tends to appear where chemotherapy has been used extensively. This problem is exacerbated by the fact there are so few drugs available for the control of some parasites. Parasites utilize the same five basic resistance mechanisms that are displayed by bacteria: 1) metabolic inactivation of the drug; 2) use of efflux pumps; 3) use of alternative metabolic pathways; 4) alteration of the target; 5) elevation of the amount of target enzyme. 6.2 Other approaches to control The early success in malaria control can be attributed to the use of professional spray teams who treated the inside of huts with DDT, without any direct involvement of the infected population. However, the problem with pesticides like DDT is that they lack specificity, and as application is not always well directed there is often destruction of a wide range of insects, which may have undesirable side-effects. Further problems include the accumu-
Biological control is an active but developing area. Genetic control of insect vectors, particularly the use of irradiated sterile males, has been widely publicized, and the release of chemically sterile males has been attempted to control anopheline mosquitoes. Other similar methods include the release of closely related species within the environment in order to produce sterile hybrids. Genetically modified mosquitoes are currently being developed that are resistant to Plasmodium infection, and larvivorous fish have also been employed for mosquito control; other organisms considered for the same purpose include bacteria, fungi, nematodes and predatory insects. One of the best studied agents is the bacterium Bacillus thuringiensis; the spore or the isolated toxin from this species can be used as a very effective and specific insecticide. 6.2.2 Vaccination Where exposure to infection is likely to occur, killing the parasite as it enters the host is a sensible approach to control. There are two options available, chemoprophylaxis or vaccination. Unfortu101
Chapter 6
nately long-term chemotherapy can have adverse side-effects, and in the absence of symptoms members of the ‘at risk’ population may fail to take the treatment. Vaccination would seem to be the ideal method of parasite control, as life-long resistance may result from just a single treatment. Despite a huge amount of effort, the only successful parasite vaccines are those for the control of veterinary parasites. However, there has been some success with the development of recombinant vaccines for the control of malaria. The recent development of DNA vaccines may be of use in the control of parasites. In this method the DNA encoding an important parasite protein is injected into host cells and the foreign ‘vaccinating’ protein is synthesized in or on the surface of the cell. This intracellular foreign
102
protein enters the cell’s major histocompatibility complex (MHC) class 1 pathway resulting in a cellmediated immune response. In contrast, a protein that is extracellular enters the MHC class 2 pathway, which results primarily in an antibody or humoral response.
7 Acknowledgement The figures for this chapter originate in the copyright-free DPDx website maintained by the United States Centre for Disease Control Division of Parasitic Diseases; this source is gratefully acknowledged.
Chapter 7
Principles of microbial pathogenicity and epidemiology Peter Gilbert and David Allison 1 Introduction 2 Portals of entry 2.1 Skin 2.2 Respiratory tract 2.3 Intestinal tract 2.4 Urinogenital tract 2.5 Conjunctiva 3 Consolidation 3.1 Resistance to host defences 3.1.1 Modulation of the inflammatory response 3.1.2 Avoidance of phagocytosis 3.1.3 Survival following phagocytosis 3.1.4 Killing of phagocytes 4 Manifestation of disease 4.1 Non-invasive pathogens
1 Introduction Microorganisms are ubiquitous, and the majority of them are free-living and derive their nutrition from inert organic and inorganic materials. The association of humans with such microorganisms is generally harmonious, as the majority of those encountered are benign and, indeed, are often vital to commerce, health and a balanced ecosystem. Each of us unwittingly carries a population of bacterial cells on our skin and in our gut that outnumbers cells carrying our own genome by ten to one. In spite of this the tissues of healthy animals and plants are essentially microbe-free. This is achieved through provision of a number of non-specific defences to those tissues, and specific defences such as antibodies (see Chapters 8 and 23) that may be acquired after exposure to particular agents. Breach of these defences by microorganisms, through the expression of virulence factors and adaptation to a pathogenic mode of life, or following disease, accidental trauma, catheterization or implantation of
5
6 7 8
4.2 Partially invasive pathogens 4.3 Invasive pathogens 4.3.1 Active spread 4.3.2 Passive spread Damage to tissues 5.1 Direct damage 5.1.1 Specific effects 5.1.2 Non-specific effects 5.2 Indirect damage Recovery from infection: exit of microorganisms Epidemiology of infectious disease Further reading
medical devices may lead to the establishment of microbial infections. The ability of bacteria and fungi to establish infections of plants, animals and man varies considerably. Some are rarely, if ever, isolated from infected tissues, while opportunist pathogens (e.g. Pseudomonas aeruginosa) can establish themselves only in compromised individuals. Only a few species of bacteria may be regarded as obligate pathogens, for which animals or plants are the only reservoirs for their existence (e.g. Neisseria gonorrhoeae). Viruses (Chapter 5) on the other hand must parasitize host cells in order to replicate and are therefore inevitably associated with disease. Even among the viruses and obligate bacterial pathogens the degree of virulence varies, in that some are able to co-exist with the host without causing overt disease (e.g. staphylococci), while others will always cause some detriment to the host organism. Organisms such as these invariably produce their effects, directly or indirectly, by actively growing on or in the host tissues. 103
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104
Food Water water
Swimming Eye drops
Conjunctiva
Air
Sputum
Upper respiratory tract Lower respiratory tract Alimentary tract
Other groups of microorganisms may cause disease through ingestion, by the victim, of substances (toxins) produced during microbial growth on foods (e.g. Clostridium botulinum, botulism; Bacillus cereus, vomiting). The organisms themselves do not have to survive and grow in the victim for the effects of the toxin to be felt. Whether such organisms should be regarded as pathogenic is debatable, but they must be considered in any account of microbial pathogenicity. Animals and plants constantly interact with bacteria present within their environment. For an infection to develop, such microorganisms must remain associated with the host and increase their numbers more rapidly than they can either be eliminated or killed. This balance relates to the ability of the bacterium to mobilize nutrients and multiply in the face of innate defences and a developing immune response by the now compromised host. The greater the number of bacterial cells associated with the initial challenge to the host, the greater will be the chance of a successful colonization. If the pathogen does not arrive at its ‘portal of entry’ to the body or directly at its target tissues in sufficient number, then an infection will not ensue. The minimum number of viable microorganisms that is required to cause infection and thereby disease is called the ‘minimum infective number’ (MIN). The MIN varies markedly between the various pathogens and is also affected by the general health and immune status of the individual host. The course of an infection can be considered as a sequence of separate events that includes initial contact with the pathogen, its consolidation and spread between and within organs and its eventual elimination (Fig. 7.1). Growth and consolidation of the microorganisms at the portal of entry commonly involves the formation of a microcolony (biofilm, see Chapter 3). Biofilms and microcolonies are collections of microorganisms that are attached to surfaces and enveloped within exopolymer matrices (glycocalyx) composed of polysaccharides, glycoproteins and/or proteins. Growth within the matrix not only protects the pathogens against opsonization and phagocytosis by the host but also modulates their micro-environment and reduces the effectiveness of many antibiotics. The localized high cell densities present within the biofilm com-
Latency
Soil, contact
Blood
Skin Viscera CNS Fetus
Insects Trauma
Crusts Scabs Skin-scales
Urinary genital tract
Urine, Semen
Sexual contact
Faeces Fig. 7.1 Routes of infection, spread and transmission of
disease.
munities also initiate production, by the colonizing organism, of extracellular virulence factors such as toxins, proteases and siderophores (low molecular weight ligands responsible for the solubilization and transport of iron (III) in microbial cells). These help combat the host’s innate defences and also promote the acquisition of nutrients by the pathogen. Viruses are incapable of growing extracellularly and must therefore rapidly gain entry to cells, generally epithelial in nature, at their initial site of entry. Once internalized, in the non-immune host, they are to a large extent protected against the nonspecific host defences. Following these initial consolidation events, the organisms may expand into surrounding tissues and/or disperse, via the blood, plasma, lymph or nerves, to distant tissues in order to establish secondary sites of infection or to consolidate further. While in some instances the microorganism is able to colonize the host indefinitely and remain viable for many years (e.g. chicken pox, cold sores) more generally it succumbs to the heightened defences of the host, and in order to sur-
Principles of microbial pathogenicity and epidemiology
vive must either infect other individuals or survive in the general environment.
2 Portals of entry 2.1 Skin The part of the body that is most widely exposed to microorganisms is the skin. Intact skin is usually impervious to microorganisms and its surface is of acid pH and contains relatively few nutrients that are favourable for microbial growth. The vast majority of organisms falling onto the skin surface will die, while the survivors must compete with the commensal microflora for nutrients in order to grow. These commensals, which include coryneform bacteria, staphylococci and yeasts, derive nutrients from compounds like urea, hormones (e.g. testosterone) and fatty acids found in the apocrine and epocrine secretions. Such organisms are highly adapted to growth in this environment and will normally prevent the establishment of chance contaminants of the skin. Infections of the skin itself, such as ringworm (Trichophyton mentagrophytes) and warts, rarely, if ever, involve penetration of the epidermis. Infection can, however, occur through the skin following trauma such as burns, cuts and abrasions and, in some instances, through insect or animal bites or the injection of contaminated medicines. In recent years extensive use of intravascular and extravascular medical devices and implants has led to an increase in the occurrence of hospital-acquired infection. Commonly these infections involve the growth of skin commensals such as Staphylococcus epidermidis when associated with devices that penetrate the skin barrier. The organism grows as an adhesive biofilm upon the surfaces of the device, and infection arises either from contamination of the device during its implantation or by growth along it of the organism from the skin. In such instances the biofilm sheds bacterial cells to the body and may give rise to a bacteraemia (the presence of bacteria in the blood). The weak spots, or Achilles heels, of the body occur where the skin ends and mucous epithelial tissues begin (mouth, anus, eyes, ears, nose and urinogenital tract). These mucous membranes present a
much more favourable environment for microbial growth than the skin, in that they are warm, moist and rich in nutrients. Such membranes, nevertheless, possess certain characteristics that allow them to resist infection. The majority, for example, possess their own highly adapted commensal microflora that must be displaced by any invading organisms. The resident flora varies greatly between different sites of the body, but is usually common to particular host species. Each site can be additionally protected by physicochemical barriers such as extreme acid pH in the stomach, the presence of freely circulating non-specific antibodies and/or opsonins, and/or by macrophages and phagocytes (see Chapter 8). All infections start from contact between these tissues and the potential pathogen. Contact may be direct, from an infected individual to a healthy one; or indirect, and may involve inanimate vectors such as soil, food, drink, air and airborne particles. These may directly contact the body or be ingested or inhaled or enter wounds via infected bed linen and clothing. Indirect contact may also involve animal vector intermediates (carriers). 2.2 Respiratory tract Air contains a large amount of suspended organic matter and, in enclosed occupied spaces, may hold up to 1000 microorganisms/m3. Almost all of these airborne organisms are non-pathogenic bacteria and fungi, of which the average person would inhale approximately 10 000 per day. The respiratory tract is protected against this assault by a mucociliary blanket that envelops the upper respiratory tract and nasal cavity. Both present a tortuous path down which microbial particles travel and inevitably impact upon these surfaces to become trapped within an enveloping blanket of mucus. Beating cilia move the mucus coating to the back of the throat where it, together with adherent particles, is swallowed. The alveolar regions of the lower respiratory tract have additional protection in the form of alveolar macrophages. To be successful, a pathogen must avoid being trapped in the mucus and swallowed, and if deposited in the alveolar sacs must avoid engulfment by macrophages or resist subsequent digestion by them. The possession of 105
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surface adhesins, specific for epithelial receptors, aids attachment of the invading microorganism and avoidance of removal by the mucociliary blanket. Other strategies include the export of ciliostatic toxins (i.e. Corynebacterium diphtheriae) that paralyse the cilial bed. As the primary defence of the respiratory tract is the mucociliary blanket, it is easy to envisage how infection with respiratory viruses (i.e. influenza virus kills respiratory epithelia) or the chronic inhalation of tobacco smoke (increases mucin production and decreases the proportion of ciliated epithelial cells) increases the susceptibility of individuals to infection. 2.3 Intestinal tract The intestinal tract must contend with whatever it is given in terms of food and drink. The extreme acidity and presence of digestive enzymes in the stomach will kill many of the bacteria challenging it, and the gastrointestinal tract carries its own commensal flora of yeast and lactobacilli that afford protection by, for example, competing with potential pathogens like Helicobacter pylori. Bile salts are mixed with the semi-digested solids exiting from the stomach into the small intestine. These salts not only neutralize the stomach acids but also contain surfactants that are able to solubilize the outer membrane of many Gram-negative bacteria. As a consequence, the small intestine is generally free from microorganisms and can be readily colonized by suitably adapted pathogens. The lower gut on the other hand is highly populated by commensal microorganisms (1011 g-1 gut tissue) that are often associated with the intestinal wall, either embedded in layers of protective mucus or attached directly to the epithelial cells. The pathogenicity of incoming bacteria and viruses depends upon their ability to survive passage through the stomach and duodenum and upon their capacity for attachment to, or penetration of, the gut wall, despite competition from the commensal flora and the presence of secretory antibodies (Chapter 8). 2.4 Urinogenital tract In healthy individuals, the bladder, ureters and urethra are sterile, and sterile urine constantly flushes 106
the urinary tract. Organisms invading the urinary tract must avoid being detached from the epithelial surfaces and washed out during urination. In the male, as the urethra is long (c. 20 cm), bacteria must be introduced directly into the bladder, possibly through catheterization. In the female, the urethra is much shorter (c. 5 cm) and is more readily traversed by microorganisms that are normally resident within the vaginal vault. Bladder infections are therefore much more common in the female. Spread of the infection from the bladder to the kidneys can easily occur through reflux of urine into the ureter. As for the implantation of devices across the skin barrier (above), long-term catheterization of the bladder will promote the occurrence of bacteriuria (the presence of bacteria in the urine) with all of the associated complications. Lactic acid in the vagina gives it an acidic pH (5.0); this, together with other products of metabolism inhibits colonization by most bacteria, except some lactobacilli that constitute the commensal flora. Other types of bacteria are unable to establish themselves in the vagina unless they have become extremely specialized. These species of microorganism tend to be associated with sexually transmitted infections. 2.5 Conjunctiva The conjunctiva is usually free of microorganisms and protected by the continuous flow of secretions from lachrymal and other glands, and by frequent mechanical cleansing of its surface by the eyelid periphery during blinking. Lachrymal fluids contain a number of inhibitory compounds, together with lysozyme, that can enzymically degrade the peptidoglycan of Gram-positive bacteria such as staphylococci. Damage to the conjunctiva, caused through mechanical abrasion or reductions in tear flow, will increase microbial adhesion and allow colonization by opportunist pathogens. The likelihood of infection is thus promoted by the use of soft and hard contact lenses, physical damage, exposure to chemicals, or damage and infection of the eyelid border (blepharitis).
Principles of microbial pathogenicity and epidemiology
3 Consolidation To be successful, a pathogen must be able to survive at its initial portal of entry, frequently in competition with the commensal flora and generally subject to the attention of macrophages and wandering white blood cells. Such survival invariably requires the organism to attach itself firmly to the epithelial surface. This attachment must be highly specific in order to displace the commensal microflora, and subsequently governs the course of an infection. Attachment can be mediated through provision, on the bacterial surface, of adhesive substances such as mucopeptide and mucopolysaccharide slime layers, fimbriae (Chapter 3), pili (Chapter 3) and agglutinins (Chapter 8). These are often highly specific in their binding characteristics, differentiating, for example, between the tips and bases of villi in the large bowel and the epithelial cells of the upper, mid and lower gut. Secretory antibodies, which are directed against such adhesions, block the initial attachment of the organism and thereby confer resistance to infection. The outcome of the encounter between the tissues and potential pathogens is governed by the ability of the microorganism to multiply at a faster rate than it is removed from those tissues. Factors that influence this are the organism’s rate of growth, the initial number arriving at the site and their ability to resist the efforts of the host tissues at removing/starving/killing them. The definition of virulence for pathogenic microorganisms must therefore relate to the minimum number of cells required to initiate an infection (MIN). This will vary between individuals, but will invariably be lower in compromised hosts such as those who are catheterized, diabetics, smokers and cystic fibrotics, and those suffering trauma such as malnutrition, chronic infection or physical damage. A number of the individual factors that contribute towards virulence are discussed below. 3.1 Resistance to host defences Most bacterial infections confine themselves to the surface of epithelial tissue (e.g. Bordetella pertussis, Corynebacterium diphtheriae, Vibrio cholerae). This is, to a large extent, a reflection of their inabili-
ty to combat that host’s deeper defences. Survival at these sites is largely due to firm attachment to the epithelial cells. Such organisms manifest disease through the production and release of toxins (see below). Other groups of organisms regularly establish systemic infections after traversing the epithelial surfaces (e.g. Brucella abortus, Salmonella typhi, Streptococcus pyogenes). This property is associated with their ability either to gain entry into susceptible cells and thereby enjoy protection from the body’s defences, or to be phagocytosed by macrophages or polymorphs yet resist their lethal action and multiply within them. Other organisms are able to multiply and grow freely in the body’s extracellular fluids. Microorganisms have evolved a number of different strategies that allow them to suppress the host’s normal defences and thereby survive in the tissues. 3.1.1 Modulation of the inflammatory response Growth of microorganisms releases cellular products into their surrounding medium, many of which cause non-specific inflammation associated with dilatation of blood vessels. This increases capillary flow and access of phagocytes to the infected site. Increased lymphatic flow from the inflamed tissues carries the organisms to lymph nodes where further antimicrobial and immune forces come into play (Chapter 8). Many of the substances that are released by microorganisms in this fashion are chemotactic towards polymorphs that tend, therefore, to become concentrated at the site of infection: this is in addition to the inflammation and white blood cell accumulation that is associated with antibody binding and complement fixation (Chapter 8). Many organisms have adapted mechanisms that allow them to overcome these initial defences. Thus, virulent strains of Staphylococcus aureus produce a mucopeptide (peptidoglycan), which suppresses early inflammatory oedema, and related factors that suppress the chemotaxis of polymorphs. 3.1.2 Avoidance of phagocytosis Resistance to phagocytosis is sometimes associated 107
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with specific components of the cell wall and/or with the presence of capsules surrounding the cell wall. Classic examples of these are the M-proteins of the streptococci and the polysaccharide capsules of the pneumococci. The acidic polysaccharide Kantigens of Escherichia coli and Salmonella typhi behave similarly, in that (i) they can mediate attachment to the intestinal epithelial cells, and (ii) they render phagocytosis more difficult. Generally, possession of an extracellular capsule will reduce the likelihood of phagocytosis. Microorganisms are more readily phagocytosed when coated with antibody (opsonized). This is due to the presence on the white blood cells of receptors for the Fc fragment of IgM and IgG (discussed in Chapter 8). Avoidance of opsonization will clearly enhance the chances of survival of a particular pathogen. A substance called protein A is released from actively growing strains of Staph. aureus, which acts by non-specific binding to IgG at the Fc region (see also Chapter 8), at sites both close to, and remote from, the bacterial surface. This blocks the Fc region of bound antibody masking it from phagocytes. Protein A–IgG complexes remote from the infection site will also bind complement, thereby depleting it from the plasma and negating its actions near to the infection site. 3.1.3 Survival following phagocytosis Death of microorganisms following phagocytosis can be avoided if the microorganisms are not exposed to the killing and digestion processes within the phagocyte. This is possible if fusion of the lysosomes with phagocytic vacuoles can be prevented. Such a strategy is employed by virulent Mycobacterium tuberculosis, although the precise mechanism is unknown. Other bacteria seem able to grow within the vacuoles despite lysosomal fusion (Listeria monocytogenes, Sal. typhi). This can be attributed to cell wall components that prevent access of the lysosomal substances to the bacterial membranes (e.g. Brucella abortus, mycobacteria) or to the production of extracellular catalase which neutralizes the hydrogen peroxide liberated in the vacuole (e.g. staphylococci). If microorganisms are able to survive and grow within phagocytes, they will escape many of the 108
other body defences such as the lymph nodes, and be distributed around the body. As the lifespan of phagocytes is relatively short then such bacteria will eventually be delivered to the liver and gastrointestinal tract where they are ‘re-cycled’. 3.1.4 Killing of phagocytes An alternative strategy is for the microorganism to kill the phagocyte. This can be achieved by the production of leucocidins (e.g. staphylococci, streptococci) which promote the discharge of lysosomal substances into the cytoplasm of the phagocyte rather than into the vacuole, thus directing the phagocyte’s lethal activity towards itself.
4 Manifestation of disease Once established, the course of a bacterial infection can proceed in a number of ways. These can be related to the relative ability of the organism to penetrate and invade surrounding tissues and organs. The vast majority of pathogens, being unable to combat the defences of the deeper tissues, consolidate further on the epithelial surface. Others, which include the majority of viruses, penetrate the epithelial layers, but no further, and can be regarded as partially invasive. A small group of pathogens are fully invasive. These permeate the subepithelial tissues and are circulated around the body to initiate secondary sites of infection remote from the initial portal of entry (Fig. 7.1). Other groups of organisms may cause disease through ingestion by the victim of substances produced during microbial growth on foods. Such diseases may be regarded as intoxications rather than as infections and are considered later (section 5.1.1). Treatment in these cases is usually an alleviation of the harmful effects of the toxin rather than elimination of the pathogen from the body. 4.1 Non-invasive pathogens Bordetella pertussis (the aetiological agent of whooping cough) is probably the best described of these pathogens. This organism is inhaled and rapidly localizes on the mucociliary blanket of the
Principles of microbial pathogenicity and epidemiology
lower respiratory tract. This localization is very selective and is thought to involve agglutinins on the organism’s surface. Toxins produced by the organism inhibit ciliary movement of the epithelial surface and thereby prevent removal of the bacterial cells to the gut. A high molecular weight exotoxin is also produced during the growth of the organism which, being of limited diffusibility, pervades the subepithelial tissues to produce inflammation and necrosis. C. diphtheriae (the causal organism of diphtheria) behaves similarly, attaching itself to the epithelial cells of the respiratory tract. This organism produces a low molecular weight, diffusible toxin that enters the blood circulation and brings about a generalized toxaemia. In the gut, many pathogens adhere to the gut wall and produce their effect via toxins that pervade the surrounding gut wall or enter the systemic circulation. Vibrio cholerae and some enteropathic E. coli strains localize on the gut wall and produce toxins that increase vascular permeability. The end result is a hypersecretion of isotonic fluids into the gut lumen, acute diarrhoea and, as a consequence, dehydration that may be fatal in juveniles and the elderly. In all these instances, binding to epithelial cells is not essential but increases permeation of the toxin and prolongs the presence of the pathogen.
are innumerable serotypes of Salmonella, which are primarily parasites of animals but are important to humans in that they colonize farm animals such as pigs and poultry and ultimately infect foods derived from them. Salmonella food poisoning (salmonellosis), therefore, is commonly associated with inadequately cooked meats, eggs and also with cold meat products that have been incorrectly stored following contact with the uncooked product. Dependent upon the severity of the lesions induced in the gut wall by these pathogens, red blood cells and phagocytes pass into the gut lumen, along with plasma, and cause the classic ‘bloody flux’ of bacillary dysentery. Similar erosive lesions are produced by some enteropathic strains of E. coli. Viral infections such as influenza and the ‘common cold’ (in reality 300–400 different strains of rhinovirus) infect epithelial cells of the respiratory tract and nasopharynx, respectively. Release of the virus, after lysis of the host cells, is to the void rather than to subepithelial tissues. The residual uninfected epithelial cells are rapidly infected resulting in general degeneration of the tracts. Such damage not only predisposes the respiratory tract to infection with opportunist pathogens such as Neisseria meningitidis and Haemophilus influenzae but it also causes the associated fever.
4.2 Partially invasive pathogens
4.3 Invasive pathogens
Some bacteria, and the majority of viruses, are able to attach to the mucosal epithelia and then penetrate rapidly into the epithelial cells. These organisms multiply within the protective environment of the host cell, eventually killing it and inducing disease through erosion and ulceration of the mucosal epithelium. Typically, members of the genera Shigella and Salmonella utilize such mechanisms in infections of the gastrointestinal tract. These bacteria attach to the epithelial cells of the large and small intestines, respectively, and, following their entry into these cells by induced pinocytosis, multiply rapidly and penetrate laterally into adjacent epithelial cells. The mechanisms for such attachment and movement are unknown but involve a transition from a non-motile to motile phenotype. Some species of salmonellae produce, in addition, exotoxins that induce diarrhoea (section 4.1). There
Invasive pathogens either aggressively invade the tissues surrounding the primary site of infection (active spread) or are passively transported around the body in the blood, lymph, cerebrospinal, axonal or pleural fluids (passive spread). Some, especially aggressive organisms, move both passively and actively, setting up multiple, expansive secondary sites of infection in various organs. 4.3.1 Active spread Active spread of microorganisms through normal subepithelial tissues is difficult in that the gel-like nature of the intercellular materials physically inhibits bacterial movement. Induced death and lysis of the tissue cells produces, in addition, a highly viscous fluid, partly due to undenatured DNA. Physical damage, such as wounds, rapidly seal with fibrin 109
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clots, thereby reducing the effective routes for spread of opportunist pathogens. Organisms such as Strep. pyogenes, Cl. perfringens and, to some extent, the staphylococci, are able to establish themselves in tissues by virtue of their ability to produce a wide range of extracellular enzyme toxins. These are associated with killing of tissue cells, degradation of intracellular materials and mobilization of nutrients. A selection of such toxins will be considered briefly. 1 Haemolysins are produced by most of the pathogenic staphylococci and streptococci. They have a lytic effect on red blood cells, releasing ironcontaining nutrients. 2 Fibrinolysins are produced by both staphylococci (staphylokinase) and streptococci (streptokinase). These toxins indirectly activate plasminogen and so dissolve fibrin clots that the host forms around wounds and lesions to seal them. The production of fibrinolysins therefore increases the likelihood of the infection spreading. Streptokinase may be employed clinically in conjunction with streptodornase (Chapter 25) in the treatment of thrombosis. 3 Collagenases and hyaluronidases are produced by most of the aggressive invaders of tissues. These are able to dissolve collagen fibres and the hyaluronic acids that function as intercellular cements; this causes the tissues to break up and produce oedematous lesions. 4 Phospholipases are produced by organisms such as Cl. perfringens (alpha-toxin). These toxins kill tissue cells by hydrolysing the phospholipids that are present in cell membranes. 5 Amylases, peptidases and deoxyribonuclease mobilize many nutrients that are released from lysed cells. They also decrease the viscosity of fluids present at the lesion by depolymerization of their biopolymer substrates. Organisms possessing the above toxins, particularly those also possessing leucocidins, are likely to cause expanding oedematous lesions at the primary site of infection. In the case of Cl. perfringens, a soil microorganism that has become adapted to a saprophytic mode of life, infection arises from an accidental contamination of deep wounds when a process similar to that seen during the decomposition of a carcass ensues (gangrene). This organism is 110
most likely to spread through tissues when blood circulation, and therefore oxygen tension, in the affected areas is minimal. Abscesses formed by streptococci and staphylococci can be deep-seated in soft tissues or associated with infected wounds or skin lesions; they become localized through the deposition of fibrin capsules around the infection site. Fibrin deposition is partly a response of the host tissues, but is also partly a function of enzyme toxins such as coagulase. Phagocytic white blood cells can migrate into these abscesses in large numbers to produce significant quantities of pus. Such pus, often carrying the infective pathogen, might be digested by other phagocytes in the late stages of the infection or discharged to the exterior or to the capillary and lymphatic network. In the latter case, blocked capillaries might serve as sites for secondary lesions. Toxins liberated from the microorganisms during their growth in such abscesses can freely diffuse to the rest of the body to set up a generalized toxaemia. Sal. typhi, Sal. paratyphi and Sal. typhimurium are serotypes of Salmonella (section 4.2) that are not only able to penetrate into intestinal epithelial cells and produce exotoxins, but are also able to penetrate beyond into subepithelial tissues. These organisms therefore produce a characteristic systemic disease (typhoid and enteric fever), in addition to the usual symptoms of salmonellosis. Following recovery from such infection the organism is commonly found associated with the gall bladder. In this state, the recovered person will excrete the organism and become a reservoir for the infection of others. 4.3.2 Passive spread When invading microorganisms have crossed the epithelial barriers they will almost certainly be taken up with lymph in the lymphatic ducts and be delivered to filtration and immune systems at the local lymph nodes. Sometimes this serves to spread infections further around the body. Eventually, spread may occur from local to regional lymph nodes and thence to the bloodstream. Direct entry to the bloodstream from the primary portal of entry is rare and will only occur when the organism damages the blood vessels or if it is injected directly into
Principles of microbial pathogenicity and epidemiology
them. This might be the case following an insect bite or surgery. Bacteraemia such as this will often lead to secondary infections remote from the original portal of entry.
5 Damage to tissues Damage caused to the host organism through infection can be direct and related to the destructive presence of microorganisms (or to their production of toxins) in particular target organs; or it can be indirect and related to interactions of the antigenic components of the pathogen with the host’s immune system. Effects can therefore be closely related to, or remote from, the infected organ. Symptoms of the infection can in some instances be highly specific, relating to a single, precise pharmacological response to a particular toxin; or they might be non-specific and relate to the usual response of the body to particular types of trauma. Damage induced by infection will therefore be considered in these categories. 5.1 Direct damage 5.1.1 Specific effects For the host, the consequences of infection depend to a large extent upon the tissue or organ involved. Soft tissue infections of skeletal muscle are likely to be less damaging than, for instance, infections of the heart muscle and central nervous system. Infections associated with the epithelial cells that make up small blood vessels can block or rupture them to produce anoxia or necrosis in the tissues that they supply. Cell and tissue damage is generally the result of a direct local action by the microorganisms, usually concerning action at the cytoplasmic membranes. The target cells are usually phagocytes and are generally killed (e.g. by Brucella, Listeria, Mycobacterium). Interference with membrane function through the action of enzymes such as phospholipase causes the affected cells to leak. When lysosomal membranes are affected, the lysosomal enzymes disperse into the cells and tissues causing them, in turn, to autolyse. This is mediated through the vast battery of enzyme toxins available to these organisms (section 4). If these toxins are
produced in sufficient concentration they may enter the circulatory systems to produce a generalized toxaemia. During their growth, other pathogens liberate toxins that possess very precise, singular pharmacological actions. Diseases mediated in this manner include diphtheria, tetanus and scarlet fever. In diphtheria, the organism C. diphtheriae confines itself to epithelial surfaces of the nose and throat and produces a powerful toxin which affects an elongation factor involved in eukaryotic protein biosynthesis. The heart and peripheral nerves are particularly affected, resulting in myocarditis (inflammation of the myocardium) and neuritis (inflammation of a nerve). Little damage is produced at the infection site. Tetanus occurs when Cl. tetani, ubiquitous in the soil and the faeces of herbivores, contaminates wounds, especially deep puncture-type lesions. These might be the result of a minor trauma such as a splinter, or a major one such as a motor vehicle accident. At these sites, tissue necrosis, and possibly also microbial growth, reduce the oxygen tension to allow this anaerobe to multiply. Its growth is accompanied by the production of a highly potent toxin that passes up peripheral nerves and diffuses locally within the central nervous system. The toxin has a strychnine-like action and affects normal function at the synapses. As the motor nerves of the brainstem are the shortest, the cranial nerves are the first affected, with twitches of the eyes and spasms of the jaw (lockjaw). A related organism, Cl. botulinum, produces a similar toxin that may contaminate food if the organism has grown in it and if conditions are favourable for anaerobic growth. Meat pastes and pâtés are likely sources. This toxin interferes with acetylcholine release at cholinergic synapses and also acts at neuromuscular junctions. Death from this toxin eventually results from respiratory failure. Many other organisms are capable of producing intoxication following their growth on foods. Most common among these are the staphylococci and strains of Bacillus cereus. Some strains of Staph. aureus produce an enterotoxin which acts upon the vomiting centres of the brain. Nausea and vomiting therefore follow ingestion of contaminated foods 111
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and the delay between eating and vomiting varies between 1 and 6 hours depending on the amount of toxin ingested. Bacillus cereus also produces an emetic toxin but its actions are delayed and vomiting can follow up to 20 hours after ingestion. The latter organism is often associated with rice products and will propagate when the rice is cooked (spore activation) and subsequently reheated after a period of storage. Scarlet fever is produced following infection with certain strains of Strep. pyogenes. These organisms produce a potent toxin that causes an erythrogenic skin rash that then accompanies the more usual effects of a streptococcal infection. 5.1.2 Non-specific effects If the infective agent damages an organ and affects its functioning, this can manifest itself as a series of secondary disease features that reflect the loss of that function to the host. Thus, diabetes may result from an infection of the islets of Langerhans, paralysis or coma from infections of the central nervous system, and kidney malfunction from loss of tissue fluids and its associated hyperglycaemia. In this respect virus infections almost inevitably result in the death and lysis of the host cells. This will result in some loss of function by the target organ. Similarly, exotoxins and endotoxins can also be implicated in non-specific symptoms, even when they have welldefined pharmacological actions. Thus, a number of intestinal pathogens (e.g. V. cholerae, E. coli) produce potent exotoxins that affect vascular permeability. These generally act through adenylate cyclase, raising the intracellular levels of cyclic AMP (adenosine monophosphate). As a result of this the cells lose water and electrolytes to the surrounding medium, the gut lumen. A common consequence of these related, yet distinct, toxins is acute diarrhoea and haemo-concentration. Kidney malfunction might well follow and in severe cases lead to death. Symptomologically there is little difference between these conditions and the food poisoning induced by ingestion of staphylococcal enterotoxin. The latter toxin is formed by the organisms during their growth on infected food substances and is absorbed actively from the gut. It acts, not at the epithelial cells of the gut, but at the 112
vomiting centre of the central nervous system causing nausea, vomiting and diarrhoea within 6 hours (above). 5.2 Indirect damage Inflammatory materials are released not only from necrotic cells but also directly from the infective agent. Endotoxins are derived from the parts that form the bacterial cell rather than being deliberately exported cellular products. Thus, during the growth and autolysis of Gram-negative bacteria components of their cell envelopes, such as lipopolysaccharide (see Chapter 3) are shed to the environment. Endotoxins tend to be less toxic than exotoxins and have much less precise pharmacological actions. Indeed, it is not always clear to what extent these can be related to actions by the host or by the pathogen. Reactions include local inflammation, elevations in body temperature, aching joints, and head and kidney pain. Inflammation causes swelling, pain and reddening of the tissues, and sometimes loss of function of the organs affected. These reactions may sometimes be the major sign and symptom of the disease. While various toxic effects have been attributed to these endotoxins, their role in the establishment of the infection, if any, remains unclear. The most notable effect of these materials is their ability to induce a high body temperature (pyrogenicity) (Chapters 3 and 19). The pyrogenic effect of lipopolysaccharide relates to the action of the lipidA component directly upon the hypothalamus and also to its direct action on macrophages and phagocytes. Elevation of body temperature follows within 1–2 hours. In infections such as meningitis the administration of antibiotics may cause such a release of pyrogen that the resultant inflammation and fever is fatal. In such instances antibiotics are co-administered with steroids to counter this effect. The pyrogenic effects of lipid-A are unaffected by moist heat treatment (autoclave). Growth of Gramnegative organisms such as Pseudomonas aeruginosa in stored water destined for use in terminally sterilized products will cause the final product to be pyrogenic. Processes for the destruction of pyrogen associated with glassware, and tests for the absence of pyrogen in water and product therefore form an
Principles of microbial pathogenicity and epidemiology
important part of parenteral drug manufacture (Chapter 19). Many microorganisms minimize the effects of the host’s defence system against them by mimicking the antigenic structure of the host tissue. The eventual immunological response of the host to infection then leads to the autoimmune destruction of itself. Thus, infections with Mycoplasma pneumoniae can lead to production of antibody against normal group O erythrocytes with concomitant haemolytic anaemia. If antigen released from the infective agent is soluble, antigen–antibody complexes are produced. When antibody is present at a concentration equal to or greater than the antigen, such as in the case of an immune host, these complexes precipitate and are removed by macrophages present in the lymph nodes. When antigen is present in excess, the complexes, being small, continue to circulate in the blood and are eventually filtered off by the kidneys, becoming lodged in kidney glomeruli and in the joints. Localized inflammatory responses in the kidneys are sometimes then initiated by the complement system (Chapter 8). Eventually the filtering function of the kidneys becomes impaired, producing symptoms of chronic glomerulonephritis.
6 Recovery from infection: exit of microorganisms The primary requirement for recovery is that multiplication of the infective agent is brought under control, that it ceases to spread around the body and that the damaging consequences of its presence are arrested and repaired. Such control is brought about by the combined function of the phagocytic, immune and complement systems. A successful pathogen will not seriously debilitate its host; rather, the continued existence of the host must be ensured in order to maximize the dissemination of the pathogen within the host population. From the microorganism’s perspective the ideal situation is where it can persist permanently within the host and be constantly released to the environment. While this is the case for a number of virus infections (chickenpox, herpes) and for some bacterial ones, it is not common. Generally, recovery from
infection is accompanied by complete destruction of the organism and restoration of a sterile tissue. Alternatively, the organism might return to a commensal relationship with the host on the epithelial and skin surface. Where the infective agent is an obligate pathogen, a means must exist for it to infect other individuals before its eradication from the host organism. The route of exit is commonly related to the original portal of entry (Fig. 7.1). Thus, pathogens of the intestinal tract are liberated in the faeces and might easily contaminate food and drinking water. Infective agents of the respiratory tract might be exhaled during coughing, sneezing or talking, survive in the associated water droplets and infect, through inhalation, nearby individuals. Infective agents transmitted by insect and animal vectors may be spread through those same vectors, the insects/ animals having been themselves infected by the diseased host. For some ‘fragile’ organisms (e.g. N. gonorrhoeae, Treponema pallidum), direct contact transmission is the only means of spread between individual hosts. In these cases, intimate contact between epithelial membranes, such as occurs during sexual contact, is required for transfer to occur. For opportunist pathogens, such as those associated with wound infections, transfer is less important because the pathogenic role is minor. Rather, the natural habitat of the organism serves as a constant reservoir for infection.
7 Epidemiology of infectious disease Spread of a microbial disease through a population of individuals can be considered as vertical (transferred from one generation to another) or horizontal (transfer occurring within genetically unrelated groups). The latter can be divided into common source outbreaks, relating to infection of a number of susceptible individuals from a single reservoir of the infective agent (i.e. infected foods), or propagated source outbreaks, where each individual provides a new source for the infection of others. Common source outbreaks are characterized by a sharp onset of reported cases over the course of a single incubation period and relate to a common experience of the infected individuals. The number of 113
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cases will persist until the source of the infection is removed. If the source of the infection remains (i.e. a reservoir of insect vectors) then the disease becomes endemic to the exposed population with a constant rate of infection. Propagated source outbreaks, on the other hand, show a gradual increase in reported cases over a number of incubation periods and eventually decline when the majority of susceptible individuals in the population have been affected. Factors that contribute to propagated outbreaks of infectious disease are the infectivity of the agent (I), the population density (P) and the numbers of susceptible individuals in it (F). The likelihood of an epidemic occurring is given by the product of these three factors (i.e. FIP). Increases in any one of them might initiate an outbreak of the disease in epidemic proportions. Thus, reported cases of particular diseases show periodicity, with outbreaks of epidemic proportion occurring only when FIP exceeds certain critical threshold values, related to the infectivity of the agent. Outbreaks of measles and chickenpox therefore tend to occur annually in the late summer among children attending school for the first time. This has the effect of concentrating all susceptible individuals in one, often
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confined, space at the same time. The proportion of susceptible individuals can be reduced through rigorous vaccination programmes (Chapter 9). Provided that the susceptible population does not exceed the threshold FIP value, then herd immunity against epidemic spread of the disease will be maintained. Certain types of infectious agent (e.g. influenza virus) are able to combat herd immunity such as this through undergoing major antigenic changes. These render the majority of the population susceptible, and their occurrence is often accompanied by spread of the disease across the entire globe (pandemics).
8 Further reading Salyers, A. A. & Drew, D. D. (2001) Microbiology: Diversity, Disease and the Environment. Fitzgerald Science Press, Bethesda, MD. Smith, H. (1990) Pathogenicity and the microbe in vivo. J Gen Microbiol, 136, 377–393. Wilson, M., McNab, R. & Henderson, B. (2002) Bacterial Disease Mechanisms. Cambridge University Press, Cambridge.
Part 2
Antimicrobial Agents
Chapter 8
Basic aspects of the structure and functioning of the immune system Mark Gumbleton and James Furr 1 Introduction 1.1 Historical perspective and scope of immunology 1.2 Definitions and outline structure of the immune system 1.3 Cells of the immune system 2 The innate immune system 2.1 Innate barriers at epidermal and mucosal surfaces 2.2 Innate defence once epidermal or mucosal barriers have been compromised 2.2.1 Mononuclear phagocytic cells 2.2.2 Granulocyte cell populations 2.2.3 Phagocytosis 2.2.4 Alternative complement pathway 3 The humoral adaptive immune system 3.1 B-lymphocyte antigens 3.2 Basic structure of antibody molecule 3.3 Clonal selection and expansion 3.4 Humoral immune effector functions 3.4.1 Cognitive function on B-lymphocyte cell surface
1 Introduction 1.1 Historical perspective and scope of immunology Progress in immunological science has been driven by the need to understand and exploit the generation of immune states exemplified now by the use of modern vaccines. From almost the first recorded observations, it was recognized that persons who had contracted and recovered from certain infectious diseases were not susceptible (i.e. were immune) to the effects of the same disease when re-exposed to the infection. Thucydides, over 2500 years ago, described in detail an epidemic in Athens (which could have been typhus or plague) and noted that sufferers were ‘touched by the pitying
3.4.2 3.4.3 3.4.4 3.4.5 3.4.6 3.4.7 3.4.8
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Neutralization of antigen by secreted antibody Opsonization of antigen Mucosal immunity Antibody-dependent cell cytotoxicity (ADCC) Immediate hypersensitivity Neonatal immunity Activation of the classical complement pathway Cell-mediated adaptive immune system 4.1 T-lymphocyte antigen recognition and MHC proteins 4.2 Processing of proteins to allow peptide presentation by MHC molecules Some clinical perspectives 5.1 Transplantation rejection 5.2 Hypersensitivity Summary Further reading
care of those who had recovered because they were themselves free of apprehension, for no-one was ever attacked a second time or with a fatal result’. Since that time many attempts have been made to induce this immune state. In ancient times the process of variolation (the inoculation of live organisms of smallpox obtained from the diseased pustules of patients who were recovering from the disease) was practised extensively in India and China. The success rate was very variable and often depended on the skill of the variolator. In the late 18th century Edward Jenner, an English country doctor, observed the similarity between the pustules of smallpox and those of cowpox, a disease that affected the udders of cows. He also observed that milkmaids who had contracted cowpox by the handling of diseased udders were immune to small117
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pox. Jenner deliberately inoculated a young boy with cowpox, and after the boy’s recovery, inoculated him again with the contents of a pustule taken from a patient suffering from smallpox; the boy did not succumb to infection from this first, or any subsequent challenges, with the smallpox virus. Even though the mechanisms by which this protection against smallpox were not understood, Jenner’s work had shown proof of principle that the harmless stimulation of our adaptive immune system (see below) was capable of generating an immune state against a specific disease, and thereby provided the basis for the process we now understand as vaccination. The cowpox virus is otherwise known as the vaccinia virus and the term vaccine was introduced by Pasteur to commemorate Jenner’s work. In 1801 Jenner prophesied the eradication of smallpox by the practice of vaccination. In 1967 smallpox infected 10 million people worldwide. The World Health Organization (WHO) initiated a programme of confinement and vaccination with the aim of eradicating the disease. In Somalia in 1977 the last case of naturally acquired smallpox occurred, and in 1979 the WHO announced the total eradication of smallpox, thus fulfilling Jenner’s prophecy. Many vaccine products are now available designed to provide protection against a range of infectious diseases. Their value has been proven in national vaccine programmes leading to dramatic reductions in morbidity and mortality of such diseases as diphtheria, pertussis, mumps, measles, rubella, hepatitis A and B. Further progress in the understanding of the complex nature and functioning of the immune system has been gained through the recognition that many varied forms of pathology, beyond that of infectious disease per se, have an underlying immunological basis, including such diseases as asthma, diabetes, rheumatoid arthritis and many forms of cancer. A basic knowledge of how the immune system functions is essential for health professionals involved in understanding the nature of disease and rationalizing therapeutic strategies. This chapter aims to provide a sound overview of the structure and functioning of the immune system and impart the reader with knowledge which will serve as a platform for the study of more complex specialized texts if and when required. 118
1.2 Definitions and outline structure of the immune system The primary function of the immune system is to defend against and eliminate ‘foreign’ material, and to minimize any damage that may be caused as a result of the presence of such material. The term ‘foreign’ includes not only potentially pathogenic microorganisms but also cells recognized as ‘non-self’ and therefore foreign such as the human body’s own virally infected or otherwise transformed (e.g. cancerous) host cells. Foreign material would also include allogeneic (within species) or xenogeneic (between species) transplant tissue and therapeutic proteins administered as medicines if they arose from a different species or were of human origin but had undergone inappropriate post-translational modifications during manufacture or contained impurities. It is also possible that small organic-based drugs may form adducts with endogenous proteins leading to the generation of an immunogen. A good example of such adduct formation is that between the serum protein albumin and the glucuronide metabolite of some non-steroidal anti-inflammatory drugs. This adduct is proposed as the basis of some hypersensitivity reactions. For clarity there are a number of terms that should be defined at this point. An organism which has the ability to cause disease is termed a pathogen. The term virulence is used to indicate the degree of pathogenicity of a given strain of microorganism. Reduction in the virulence of a pathogen is termed attenuation; this can eventually result in an organism losing its virulence completely and it is then termed avirulent. An antigen is a component of the ‘foreign’ material that gives rise to the primary interaction with the body’s immune system. If the antigen elicits an immune response it may then be termed an immunogen. Within a given antigen, e.g. a protein, there will be antigenic determinants or epitopes, which actually represent the antigen recognition sites for our adaptive immune system (see below). For example, within a protein antigen, an epitope for an antibody response will comprise 5–20 amino acids that arise either as part of a linear chain or as a cluster of amino acids brought together conformationally by the folding of the protein. Antibodies
Basic aspects of the structure and functioning of the immune system
(otherwise known as immunoglobulins — Ig) are produced and secreted into biological fluids by our adaptive immune system, are widely used in in vitro diagnosis and have been investigated in therapeutics as a means of targeting drugs to specific sites in the body. A monoclonal antibody refers to an antibody nominally recognizing only a single antigen (e.g. a single protein) and within which only a single common epitope (e.g. clusters comprising a common single specific amino acid sequence or pattern) is recognized. In contrast, a polyclonal antibody refers to an antibody nominally recognizing only a single antigen but within which a number of different epitopes (e.g. clusters comprising different amino acid sequences or patterns) are recognized. The immune system is broadly considered to exhibit two forms of response: 1 The innate immune response, which is nonspecific, displays no time lag in responsiveness, and is not intrinsically affected by prior contact with infectious agent. 2 The adaptive immune response, which displays a time lag in response, involves highly specific recognition of antigen and affords the generation of immunological memory. An example of immunological memory is that provided by the generation of specific lymphocyte memory cell populations following vaccination with an antigen (e.g. diphtheria toxoid). These memory cells reside over a long term in our lymphoid tissue and permit a more rapid and pronounced protective immunological response upon future exposure to the same antigen. The adaptive immune system is further subdivided into: (a) Humoral immunity within which the effector cells are B lymphocytes and where antigen recognition occurs through interactions with antibodies. (b) Cell-mediated immunity within which the effector cells are T lymphocytes and where antigen recognition occurs through interactions of peptide antigen (presented on the surface of other cell types) with T-cell receptors (TCR) on the plasma membrane of T lymphocytes. In cell-mediated immunity the peptide antigen must be presented to T lymphocytes by other cell types in association with a class of plasma membrane molecules termed major histocompatibility complex (MHC) proteins.
1.3 Cells of the immune system A schematic overview of the cells involved in both the innate and adaptive components of the immune response is shown in Fig. 8.1. The majority of cells involved in the immune system arise from progenitor cell populations within the bone marrow. The differentiation of these progenitor cells is under the control of a variety of growth factors, e.g. granulocyte- or macrophage-colony stimulating factors (G-CSF and M-CSF, respectively) released by monocyte and macrophage cells as well as by fibroblasts and activated endothelial cells. These growth factors promote the growth and maturation of monocyte and granulocyte populations within the bone marrow before their release into the lymphoid and blood circulations. The key cells of the innate immune system include the following. (A) Mononuclear phagocytic cells which comprise the short-lived (< 8 h) monocytes in the blood circulation and which migrate into tissues and undergo further differentiation to give rise to the long-lived and key effector cell — the macrophage. (B) The granulocyte cell populations which include the neutrophil, basophil and eosinophil. (C) The mast cell which is a tissueresident cell that is triggered by tissue damage or infection to release numerous initiating factors leading to an inflammatory response. Such factors include histamine, leukotrienes B4, C4, D4, proinflammatory cytokines (signal proteins released by leucocytes — white blood cells), e.g. TNF-a, and chemotactic substances such as interleukin-8 (IL-8). The sudden degranulation of the contents of mast cells is also responsible for the acute anaphylactic reactions to bee stings, penicillins, nuts etc. (D) The natural killer (NK) cell which has a phenotype similar to that of lymphocytes but lacks their specific recognition receptors. The NK cell exploits non-specific recognition to elicit cytotoxic actions against host cells infected with virus and those host cells that have acquired tumour cell characteristics. The lymphocyte populations also arise from bone marrow progenitor cells. The B lymphocytes mature or differentiate in the bone marrow before leaving to circulate in the blood and lymph, while T lymphocytes undergo maturation in the thymus. Antibodies mediating the effector functions of the 119
Chapter 8 Bone marrow
Blood/lymphatics Thymus
Lymphoid progenitor cell
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Neutrophil Granulocytemonocyte progenitor cell Eosinophil
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NK Natural killer cell Fig. 8.1 An overview of the cells involved in the immune response: both innate and adaptive components. The cells arise from a
pluripotent progenitor cell within the bone marrow, with their growth and differentiation controlled by numerous growth factors. The T lymphocytes differentiate in the thymus gland.
humoral immune system are produced and secreted from a differentiated B lymphocyte cell population termed plasma cells.
2 The innate immune system 2.1 Innate barriers at epidermal and mucosal surfaces Innate defence against the passage of potentially pathogenic microorganisms across epidermal and mucosal barriers involves a range of non-specific mechanisms. Commensal microorganisms upon 120
mucosal surfaces and upon membranes such as skin and conjunctiva constitute one such mechanism. These commensals are, under normal circumstances, non-pathogenic, and help prevent colonization by pathogenic strains. There are also a number of physical and chemical barriers against microbial entry, including the flow of fluid secretions from tear ducts, the urogenital tract and the skin. Many of these secretions possess bacteristatic or bactericidal activity due to their low pH or the presence of hydrolytic enzymes such as lysozyme (a peptidoglycan hydrolase). Similarly, the mucus barrier, covering mucosal surfaces such as the epithelium of the lung, serves as a false binding plat-
Basic aspects of the structure and functioning of the immune system
form for microorganisms, preventing them interacting with the underlying host cells. In the normal state the hydrated mucus barrier is efficiently cleared under the driving force of beating cilia. The serious lung infections manifest in cystic fibrosis arise because of the inability of patients to clear the bacteria-laden dehydrated mucus effectively. 2.2 Innate defence once epidermal or mucosal barriers have been compromised The function of the innate defence system against microorganisms that have penetrated into interstitial tissues and the vascular compartment relies largely upon the processes of phagocytosis (see section 2.2.3) and of activation of the alternative complement pathway (see section 2.2.4). However, the functions of the innate system when exposed to microbial infection are also critical to the recruitment and activation of cells of the adaptive immune response (see later). The main cells mediating phagocytosis are the mononuclear phagocytic cells and granulocyte cell populations; of the latter neutrophils are particularly important. For such cells to function, they must possess receptors to sense signals from their environment. In executing their effector functions they need to secrete a range of molecules that will recruit or activate other immune cells to a site of infection. Before consideration of the process of phagocytosis, an overview of the mononuclear phagocytic cell and granulocyte cell populations will be given. 2.2.1 Mononuclear phagocytic cells The mononuclear phagocytic cells include monocytes and macrophages. The monocyte comprises approximately 5% of the circulating blood leucocyte population and is a short-lived cell (circulating in blood £8 h), but which migrates into tissue to give rise to the tissue macrophage. The macrophage constitutes a long-lived, widely distributed heterogeneous population of cell types which bear different names within different tissues, such as the migrating Kupffer cell within the liver or the fixed mesangial cell within the kidney glomerulus. The mononuclear phagocytic cells secrete a wide
range of molecules too numerous to list in full here. However, these secretions include: 1 Molecules which can break down or permeabilize microbial membranes and thereby mediate extracellular killing of microorganisms, e.g. enzymes (lysozyme or cathepsin G), bactericidal reactive oxygen species and cationic proteins. 2 Cytokines which can provide innate protective antiviral (e.g. interferon-a or -b) and anti-tumour (e.g. tumour necrosis factor — TNF-a) activity against other host cells. A group of cytokines termed chemokines can also serve to chemoattract other leucocytes into an area of ongoing infection or inflammation, for example IL-8 which attracts neutrophils. Yet another group of cytokines has pro-inflammatory actions (e.g. IL-1 and TNF-a) which, among other outcomes, will lead to activation of endothelial and leucocyte cells promoting increased leucocyte extravasation into tissues and in the case of IL-1 activate T-lymphocyte populations. 3 Bioactive lipids (e.g. thromboxanes, prostaglandins and leukotrienes), which further promote the inflammatory response through actions to increase capillary vasodilation and permeability. The mononuclear phagocytic cells also possess numerous receptors that interact with their environment. These cells possess, among others: • Receptors for chemotaxis toward microorganisms, e.g. receptors for secreted bacterial peptides such as formylmethionyl peptide. • Receptors for complement proteins that serve as leucocyte activators (e.g. C3a and C5a; see section 2.2.4) or complement proteins that serve to coat (to opsonize) microorganisms (e.g. C3b). An opsonized microbial surface more readily adheres to a phagocyte membrane, with the opsonin triggering enhanced activity of the phagocyte itself. • Receptors for promoting adherence such as lectin receptors interacting with carbohydrate moieties on the surface of the microorganism, or receptors for Fc domains (non-antigen-recognition domains) of antibodies which opsonize microorganisms (e.g. the receptor for the Fc domain of IgG is Fcg), or integrin receptors for cell–cell adhesion (e.g. promoting interaction between a macrophage and T lymphocyte). 121
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• Receptors for cytokines including those involved in macrophage activation (e.g. interferon-g — IFN . -g) or limiting macrophage mobility (e.g. macrophage inhibitory factor — MIF) and hence increasing cell retention at a site of infection.
very effective generators of reactive oxygen species. Eosinophils are poor phagocytic cells and have a specialized role in the extracellular killing of parasites such as helminths, which cannot be physically phagocytosed. Basophils are non-phagocytic cells.
2.2.2 Granulocyte cell populations 2.2.3 Phagocytosis
The granulocyte cell populations include the neutrophils, basophils and eosinophils. The short-lived (~2–3 days) neutrophil is the most abundant granulocyte (comprising >90% of all circulating blood granulocytes) and is the most important in terms of phagocytosis; indeed this is the main function of the neutrophil. The receptors and secretions of the neutrophil are similar to those of the macrophage, although notably the neutrophil does not present antigen via MHC class II proteins (see later). The neutrophil is recruited to sites of tissue infection or inflammation by a neutrophil-specific chemotactic factor (IL-8) and is also chemoattracted and activated by some of the same factors described for mononuclear phagocytic cells, including complement protein C3a, bacterial formylmethionyl peptides and leukotrienes. Like macrophages, neutrophils undergo a respiratory burst and are
Macrophages and neutrophils in particular demonstrate a high capacity for the physical engulfment of particles such as microorganisms or microbial fragments from their immediate extracellular environment. This process (Fig. 8.2) comprises a number of steps. • Chemotaxis of the phagocyte toward the microorganism through signals arising from the microorganism itself (e.g. formylmethionyl peptide), signals arising from complement proteins (e.g. C3a and C5a) generated as part of the activation of the alternative complement pathway (see below), or signals due to release of inflammatory factors (e.g. leukotrienes) secreted by other leucocyte cells situated at the site of an infection. • Adherence of the microorganism to the surface of the phagocyte (step A in Fig. 8.2), involving:
Lectin adherence
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lysosome
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phagosome
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phagolysosome
R.O. species Macrophage membrane
lysozyme lactoferrin proteases DNases cationic proteins
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Phagolysosome
Fig. 8.2 Schematic of phagocytosis showing: (step A) adherence of the microorganism to the surface of the phagocyte; (step B) membrane activation of the phagocyte; (step C) enclosure of phagocytosed material within the phagosome and subsequently the phagolysosome. The mediators of the phagolysosome degradation of the microorganism are shown in the enlarged phagolysosome insert.
Basic aspects of the structure and functioning of the immune system
adhesion through lectin receptors present on the surface of the phagocyte which interact with carbohydrate moieties on the surface of the microorganism; adhesion through complement C3b receptors present on the surface of the phagocyte interacting with C3b molecules that have opsonized the surface of the microorganism; adhesion through Fc receptors which interact with the Fc domain of antibodies that have opsonized the surface of a microorganism. • Membrane activation of the phagocyte actin– myosin contractile network to extend pseudopodia around the attached microorganism (step B in Fig. 8.2). Membrane activation will also lead to the generation of a ‘respiratory burst’ by the phagocyte which involves an increase in the activity of the phagocyte membrane NADPH oxidase which converts molecular oxygen into bactericidal reactive oxygen species such as superoxide anion (•O2-), hydrogen peroxide (H2O2), and in particular hydroxyl radicals (•OH) and halogenated oxygen metabolites (HOCl-). • The enclosure of phagocytosed material, initially within a membranous vesicle termed a phagosome. Here, cationic proteins such as defensins and reactive oxygen species begin microbial membrane degradation. This is followed within minutes by fusion of the phagosome with a lysosome to form a phagolysosome, whose contents are at an acidic pH of ~5 which is optimal for the continued active breakdown of microbial structural components (step C in Fig. 8.2). 2.2.4 Alternative complement pathway The alternative complement pathway fulfils a critical role in innate immune defence. The complement system comprises at least 20 different serum proteins, many are known by the letter C and a number, e.g. C3. Many of the complement proteins are zymogens, i.e. pro-enzymes requiring proteolytic cleavage to be enzymically active themselves; some are regulatory in function. The cleavage products of complement proteins are distinguished from their precursor by the suffix ‘a’ or ‘b’, e.g. C3a and C3b, with the suffix ‘b’ generally denoting the larger fragment that stays associated with a microbial membrane, and the suffix ‘a’ generally denoting the
smaller fragment that diffuses away. The activation of the complement pathway occurs in a cascade sequence with amplification occurring at each stage, such that each individual enzyme molecule activated at one stage generates multiple activated molecules at the next. In the ‘resting’ state, in the absence of infection, the complement proteins are inactive or have only a low level of spontaneous activation. The cascade is tightly regulated by both soluble and membrane-bound associated proteins. The regulation of the complement pathway prevents inappropriate activation of the cascade (i.e. when there is no infection present) and also minimizes damage to host cells during an appropriate complement response to a microbial infection. Complement activation is normally localized to the site(s) of infection. There are three main biological functions of the alternative complement pathway. • Opsonization of microbial membranes. This involves the covalent binding of complement proteins to the surface of microbial membranes. This opsonization or coating by complement proteins promotes adherence of the opsonized microbial component(s) to the cell membranes of phagocytic cells. The complement protein C3b is a potent opsonin. • Activation of leucocytes. This involves complement proteins acting upon leucocytes, either at the site of infection or at some distance away, with the result of raising the level of functioning of the leucocytes in immune defence. For example, C3a is a potent leucocyte chemoattractant and also an activator of the respiratory burst. • Lysis of the target cell membrane. This involves a collection of complement proteins associating upon the surface of a microbial membrane to form a membrane attack complex (MAC), which leads to the formation of membrane pores and, ultimately, microbial cell lysis. Figure 8.3 shows a highly schematized view of the activation cascade for the alternative complement pathway upon a microbial membrane surface. The activation steps in the alternative pathway are also shown in Fig. 8.7, which contrasts with the activation steps in the classical complement pathway involving antibody. The pivotal protein in the alternative pathway is 123
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Factor D
C3a C3
C3b
C3bB
C3bBb
Factor B C6 C7
C9
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Factor B C5b C3 convertase
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Factor D Fig. 8.3 A highly schematized overview of the activation cascade for the alternative complement pathway on a microbial membrane surface. In the presence of a microbial membrane the C3b formed by C3 tickover deposits on the microbial membrane (step A). C3a diffuses away leading to leucocyte activation. The deposited C3b leads to the generation of a stabilized C3 convertase (step B) which, through a positive feedback loop, leads to the amplified cleavage of more C3. Some C3b associates with the C3 convertase to generate a C5 convertase (step C) which will eventually lead to the generation of an MAC.
C3 (195 kDa). Under normal circumstances (in the absence of infection) C3 is cleaved very slowly through reaction with water or trace amounts of proteolytic enzyme to give C3b and C3a. The C3b formed is susceptible to nucleophilic attack by water and is rapidly inactivated to give iC3b. The C3a is not generated in sufficient amounts to lead to leucocyte activation and is rapidly inactivated. This normal low-level cleavage of the C3 molecule is termed ‘C3 tickover’ and it provides low levels of starting material, i.e. C3b, which will be required for full activation of the alternative complement pathway in the case of a microbial infection. In the presence of a microbial membrane the C3b 124
formed by C3 tickover will be susceptible to nucleophilic attack by hydroxyl or amine groups on the membrane surface, leading to the covalent attachment of C3b to the membrane (step A in Fig. 8.3). Once C3b has attached the membrane, factor B can bind to form a molecule termed C3bB. This complex is stabilized by a soluble protein called properdin. Factor D then enzymically cleaves the bound factor B to generate a molecule termed C3bBb which is the C3 convertase of the alternative pathway (Fig. 8.3 inset). This newly generated stable C3 convertase enzymically cleaves C3 to generate further C3b and C3a molecules, leading to leucocyte activation (by
Basic aspects of the structure and functioning of the immune system
C3a) and greater deposition of C3b on the microbial membrane and hence further generation of C3 convertase molecules. In effect the microbial membrane has activated a positive feedback loop with cleavage of C3 to generate high amounts of C3b and C3a molecules. The deposited C3b not only leads to the formation of the C3 convertase but also coats the microbial membrane as an opsonin and so promotes binding to phagocyte cell membranes. Some of the deposited C3b associates with the newly formed C3 convertase to generate a complex termed C3bBb3b, which is the C5 convertase of the alternative pathway (step B in Fig. 8.3). This C5 convertase binds the complement protein C5 and cleaves it into C5a (a leucocyte activator) and C5b (an opsonin). The C5b remains associated with the membrane and acts as a platform for the sequential binding of complement proteins C6, C7, C8 (step C in Fig. 8.3). The a-chain of the C8 molecule penetrates into the microbial membrane and mediates conformational changes in the incoming C9 molecules such that C9 becomes amphipathic (simultaneously containing hydrophilic and hydrophobic groups). In this form it is capable of insertion through the microbial membrane where it mediates a polymerization process that gives the MAC. The MAC generates transmembrane channels within the microbial membrane with the osmotic pressure of the cell leading to influx of water and eventual microbial cell lysis. Differences between host cell membranes and microbial cell membranes mean that the cascade is only activated in the presence of microorganisms, so C3 tickover cannot give rise to full activation of the alternative pathway in the absence of microbial membrane. Stable deposition of a functional C3 convertase only occurs on the microbial cell surface. The differences that exist include, for example: • lipopolysaccharide or peptidoglycan on microbial membranes that promote the binding of C3b; • the high sialic acid content of host cell membranes that promotes the dissociation of any C3 convertase formed on host surfaces; • the presence of specific host cell membrane proteins that also serve a key regulatory function. Decay activating factor (DAF) or complement re-
ceptor type I (CR1) are host cell membrane proteins that serve to competitively block the binding of factor B with C3b and hence inhibit formation of a C3 convertase; they also promote disassembly of any C3 convertase formed. Membrane cofactor protein (MCP) and CR1 are further host cell membrane proteins that promote the displacement of factor B from its binding with C3b. Host cell membranes also possess a protein, CD59, which prevents the unfolding of C9 — a required step for membrane insertion to form an effective MAC.
3 The humoral adaptive immune system The humoral immune response is mediated through antibody–antigen interactions. B lymphocytes in their naïve state, unstimulated by antigen, possess antibody molecules on their cell membrane, which serve a surveillance function to recognize any invading antigen. A B lymphocyte that has bound antigen is capable of differentiating into a plasma cell which, under the influence of signals from helper T cells, produces a fuller repertoire of antibody molecules (i.e. a fuller range of antibody classes), which are then secreted from the plasma cell into the extracellular environment to bring about a range of humoral effector functions. 3.1 B-lymphocyte antigens As briefly discussed in section 1.2, a B-cell antigen is a substance or molecule specifically interacting with an antibody, and which may lead to the further production of antibody and an immunological response. Typically B-lymphocyte antigens are proteins within which the epitopes each comprise clusters of 5–20 amino acid residues in size. Blymphocyte epitopes arise most commonly from the three-dimensional folding of proteins (i.e. conformational epitopes), although they may also comprise a sequential linear sequence of amino acids within the polypeptide chain (linear epitopes). As a general rule, there is a gradient of increasing immunogenicity with increasing molecular weight of protein. Further, the higher the structural complexity of the protein or polypeptide antigen then 125
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the higher the level of immunogenicity it is likely to exhibit. Thus, a polypeptide comprising a single amino acid such as polylysine may be expected to be a weaker immunogen than a protein of equivalent molecular weight but comprising a diverse range of amino acids. Polysaccharides tend not to be good immunogens for B lymphocytes. When a polysaccharide serves as the sole immunogen, the humoral response obtained is termed ‘T-cell-independent’ because the polysaccharide does not elicit helper T-lymphocyte co-operation (see section 4). The consequences of a T-cell-independent humoral response include the lack of production of memory B-cell populations and the lack of synthesis by the plasma cell of the full range of antibody subclasses, i.e. T-cell-independent humoral responses mainly involve the production of IgM antibody. For improved immunogenicity carbohydrate antigens are conjugated to proteins which allow a more effective ‘T-cell dependent’ humoral response, i.e. one that affords the generation of memory B-cell populations and of the synthesis of the full range of antibody subclasses. This strategy is used in a number of current vaccine products, e.g. meningococcal group C conjugate vaccine comprises the capsular polysaccharide antigen of Neisseria meningitidis group C conjugated to Corynebacterium diphtheriae protein. Pure nucleic acid and lipid serve as very poor antigens.
3.2 Basic structure of antibody molecule Figure 8.4 shows an antibody monomer comprising a four-polypeptide subunit structure, where the subunits are linked through disulphide bonding. The basic monomer structure can be considered the same for all the different classes of antibody (see below) even though some may form higher order structures, e.g. IgM is a pentamer comprising five antibody monomer units. The subunits of the antibody monomer comprise two identical ‘heavy’ polypeptide chains and two identical ‘light’ polypeptide chains, with each of these containing a ‘constant’ region and a ‘variable’ region. The light chain variable regions (VL) and the heavy chain variable regions (VH) are the parts of the antibody molecule involved in antigen recognition. Specifically, antibodies produced by different B lymphocytes or plasma cells will have variable regions possessing different amino acid sequences leading to differences in antibody variable region surface conformation. At the extreme tips of the variable regions are hypervariable domains that serve the specific antigen recognition function discriminating between, for example, diphtheria toxin and tetanus toxin. The structural differences in the variable and hypervariable domains enable different antibodies to recognize different structural epitopes; this meets the needs of the immune system to combat a large and diverse range of antigens.
Fab domain Light chain VH CL Hypervariable regions
VH VH
Fc domain CH Heavy chain
CH CH
Heavy chain CH
Line of symmetry through molecule
CL VL
Fig. 8.4 An antibody monomer comprising a four-polypeptide subunit structure, where the subunits are linked through disulphide bonding. The Fab fragment is concerned with antigen recognition, while the Fc region determines the various effector functions of antibodies. A horizontal line of symmetry can be drawn through the antibody structure bisecting the molecule into a single heavy chain and a single light chain and clearly showing the bivalency in antigen recognition.
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Basic aspects of the structure and functioning of the immune system
A horizontal line of symmetry can be drawn through the antibody structure in Fig 8.4, bisecting the molecule into two equivalent halves each containing a single heavy chain and a single light chain and clearly showing the antibody monomer to possess bivalency in its ability to interact with antigen, i.e. each antibody monomer can bind two epitopes, although the epitopes bound by a single antibody must be identical. The antigen recognition domain of an antibody monomer is termed the Fab domain. The structure of the constant region of the heavy chain (CH) does not influence the antigen recognition function of the molecule but defines the different classes of antibody that are produced and hence the effector functions arising from antigen–antibody interaction; this heavy chain constant region is termed the Fc domain. An analogy that may assist visualization of the function of an antibody molecule is one that views it as a hand (Fab domain) attached to the arm (Fc domain) (Fig. 8.4). The palm of the hand (variable region) can take up different shapes to allow the fingertips (hypervariable regions) to gain a very precise interaction with an object (antigen). At the wrist (hinge region) the hand is highly flexible relative to the arm (Fc domain) to allow the hand and fingertips (Fab domain) maximum flexibility to orientate an interaction with objects (antigen). The structure of the arm (Fc domain) does not influence interaction with an object (antigen). Once the object (antigen) has interacted with the fingertips (hypervariable regions) of the hand then the arm (Fc domain) can mediate a variety of effector functions. A B lymphocyte and plasma cell can produce different classes of antibody depending on the stage of immune activation and on the intercellular signals that the B lymphocyte and plasma cell receive from other effector cells within the immune system. As stated above, the class of antibody is determined by the structure of the Fc domain and the different classes of antibodies possess different effector functions. The basic classes of antibodies are: IgM (heavy chain constant region defined as m); IgA (heavy chain constant region defined as a); IgD (heavy chain constant region defined as d); IgG (heavy chain constant region defined as g) and IgE (heavy chain constant region defined as e). The different classes of antibody can be remembered using
the acronym MADGE. In addition to the heavy chain constant region classes, there are two light chain constant region classes, k and l; however, these do not mediate different antibody effector functions. Each B lymphocyte and the plasma cell that derives from it is capable of producing all the different antibody classes. However, all the antibody classes produced by a single B lymphocyte and its derived plasma cell will recognize only a single epitope, i.e. recognize only a single specific set of chemical features within a sequence or pattern of amino acid residues. In other words, all antibodies produced by a single B lymphocyte, and its derived plasma cell, possess the same Fab domain recognizing the same antigenic determinant but clearly may possess different Fc domains capable of mediating different effector functions. Thus the same epitope can stimulate various different forms mediated via the IgM, IgA, IgD, IgG, IgE classes of humoral immune attack. Within the antibody pool it is estimated that there are approximately 109 different epitope recognition specificities, sufficient to cover the range of pathogens likely to be encountered in life. This enormous diversity in antigen recognition is due to the amino acid sequence diversity in the variable and hypervariable domains of the antibody molecule. However, this large diversity cannot result from the presence of an equivalent number of separate protein coding genes; the human genome project has estimated there to be only approximately 30 000 protein coding genes. Rather, the clonal diversity in antigen recognition is due in the main to a process termed gene rearrangement, which occurs in each B lymphocyte during maturation in the bone marrow. For example, the DNA coding for a single heavy chain molecule will result from the splicing together of genes from four separate regions termed a Variable region gene, a Diversity region gene, a Joining region gene and a Constant region gene. There are approximately 100 V genes, 25 D genes and 50 J genes. Gene rearrangement will allow combinatorial freedom for any V, D and J genes to splice together providing a large number of VDJ combined gene product permutations and hence diversity in antigen recognition. Inaccurate splicing together of the regional genes at the V–D and D–J 127
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junctions further increases diversity, as does the process of random nucleotide insertion. The Constant region genes will dictate the different classes of antibody and not the antigen recognition specificity. An additional process which occurs in a Blymphocyte memory cell population while it resides within the lymphoid tissue is that of somatic mutation, in which only very slight changes in antibody Fab domains occur through single base mutations. Sometimes these mutations prove advantageous by increasing the affinity of an antibody to the same original epitope. Under these circumstances the antibody clone with the highest binding affinity to the original target epitope will proliferate and dominate. The light chain gene also comprises V, J and C regions and the V and J genes undergo a similar rearrangement to that described for the heavy chain, and hence further add to diversity. The heavy chain and light chain polypeptides are joined together via disulphide bond formation following protein synthesis of the individual heavy and light chains. In summary, all antibodies produced by a single B lymphocyte and its derived plasma cell are ‘programmed’ to recognize only a single antigen recognition feature determined by the recombination pattern of the V, D and J genes (heavy chain) and the V and J genes (light chain). The class of antibody is determined by further excisions within the DNA to allow the same VDJ gene combination to lie next to a different C gene, which codes for the structure of the antibody constant region and therefore determines antibody class. The five C gene classes are m, a, d, g and e, although various subclasses also exist. Antibody class switching is not a random process but one that is regulated by helper T-lymphocyte cytokine secretions. 3.3 Clonal selection and expansion Within the body there may exist at any one time only a handful of naïve B lymphocytes capable of recognizing the same epitope. The meeting of an antigen and a naïve B lymphocyte capable of recognizing an epitope within the antigen occurs through the delivery of antigen to lymphoid tissues of the spleen, lymph nodes and local lymphoid tissue within mucosal surfaces (mucosal associated lymphoid tissue, MALT) and skin (SALT). This lym128
phoid tissue is rich in lymphocytes. Further, a proportion of B lymphocytes will always be recirculating from the lymphoid tissue through the lymph and blood circulations and so able to encounter circulating antigen. Antigen will be specifically recognized by IgM molecules present on the surface of the naïve B lymphocyte. Following this antigen-driven selection of a specific B-lymphocyte clone, the clone will undergo repeated cell divisions. Some of the daughter cells will differentiate into short-lived (2–3 days) plasma cells able to secrete antibody of different classes to combat the initial primary antigen exposure. Other clonal daughter cells will become longlived B-lymphocyte memory cells populating the lymphoid tissue and spreading around the body through the lymph and blood circulations. These cells will provide ‘immunological memory’ able to generate a more rapid and pronounced secondary response upon subsequent exposure to the original antigen (Fig. 8.5). This process of clonal selection and expansion to form memory cell populations is the basis of vaccination. The initial introduction of antigen gives rise to a primary response (Fig. 8.6) in which there is a significant latent period before increased serum antibody levels are observed; the main antibody response is IgM production, although some IgG is
B
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Selective activation of naïve B-lymphocyte Clonal proliferation
B
B
B
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B
B
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Memory cells Secreted antibody Fig. 8.5 Clonal proliferation of B lymphocytes. Following antigen-driven selection of a specific B-lymphocyte clone, it will undergo repeated cell divisions to give effector cell populations and memory cell populations.
Basic aspects of the structure and functioning of the immune system
Ab titer
Total Ab
1º Ag
IgM Ab
IgG Ab
2º Ag
Latent period 7-20 days Latent period 5-7 days Days after immunization Fig. 8.6 Primary and secondary responses to antigen.
A primary response of the humoral system involves a significant latent period before elevated serum antibody levels are seen; the major serum antibody generated is IgM. Memory cell populations provide the basis of the secondary response, which displays a significant reduction in the latency period to achieve elevated serum antibody. The antibody serum levels are greater than in the primary response and involve mainly IgG.
also synthesized and secreted. Upon re-exposure to the same antigen a secondary response is elicited. The features of the secondary response include: a reduced latent period between antigen challenge and increases in serum antibody (e.g. latent period of 5–7 days for the secondary response versus 7– 20 days for the primary response); an antibody response dominated by IgG which is more pronounced with higher serum levels achieved. In the absence of helper T lymphocyte involvement (Tcell-independent humoral responses, e.g. where antigen is carbohydrate alone) B-lymphocyte memory cell populations are not produced, and antibody class switching is restricted. Hence, under these circumstances, the primary and secondary antibody responses to antigen challenge are essentially indistinguishable and exhibit a prolonged latent period, relatively low levels of serum antibody produced, and IgM as the main serum antibody. 3.4 Humoral immune effector functions The humoral immune response is mediated by the
initial antibody–antigen interaction, but with the different antibody classes offering a range of effector functions. The effector functions of antibodies include those described below. 3.4.1 Cognitive function on B-lymphocyte cell surface Antibody on the surface of naïve or memory B lymphocytes serves to recognize and bind specific antigen; IgM serves this main cognitive function. It exists as a pentamer of five monomer units with an antigen valency of 10 and is extremely efficient at binding antigen. IgD appears to function mainly on the surface of B lymphocytes and may also contribute to cognition in some way. 3.4.2 Neutralization of antigen by secreted antibody Secreted antibody, in particular IgG, IgA and IgM, can bind antigen and sterically hinder the interaction of toxins, viruses, bacteria, etc. with host cell surfaces. In the circulatory and interstitial fluids IgG (which exists as a monomer with an antigen valency of 2) is the main antibody that fulfils this role in the secondary response, while IgM is the main antibody produced in the primary response. IgA has specific roles in mucosal immunity. 3.4.3 Opsonization of antigen Secreted antibody, in particular IgG, opsonizes antigenic material and in doing so promotes association (e.g. through Fcg receptors) of the antigenic material with phagocyte membranes. Occupancy of the Fc receptor by the antibody also serves to activate a phagocyte’s killing mechanisms. 3.4.4 Mucosal immunity Mucosal immunity involves the interaction of antibody with antigen at mucosal surfaces such as those of the gastrointestinal tract, lung or urogenital tract. The major antibody of the mucosal lining fluid is IgA, which exists as a dimer of two monomer units (antigen valency of 4). IgA is actively secreted across mucosal epithelium into the lining fluid; it 129
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will neutralize antigen and may also serve as an opsonin. IgA is also present in secretions such as tears, saliva, etc. but it has a limited role in systemic immunity. 3.4.5 Antibody-dependent cell cytotoxicity (ADCC) Through specific binding to antigen upon the surface of membranes perceived as ‘foreign’, e.g. microbial cells or host cells virally infected or otherwise transformed, antibody can direct (through its Fc domains) the close association of ‘killing’ cells, such as neutrophils, eosinophils, NK cells and even cytotoxic T lymphocytes, with the ‘foreign’ membrane. This close association depends upon the antibody’s Fc domain binding to the respective Fc receptor present on the surface membrane of the ‘killing’ cell. The release of cytotoxic molecules into the extracellular environment in close proximity to the ‘foreign’ cell enables the efficient and targeted release of cytotoxic molecules. IgG is the main antibody of systemic body fluids and is an important mediator of ADCC, although IgE and IgA may undertake this role in certain circumstances, e.g. against certain parasites IgE directs ADCC mediated by eosinophils. 3.4.6 Immediate hypersensitivity Mast cells express high affinity receptors (Fce) that bind the Fc domain of IgE antibodies. In the absence of antigen these receptors are occupied by the IgE monomer (antigen valency of 2) secreted previously from plasma cells. In this circumstance the IgE molecules are serving a cognitive function which, upon appropriate antigen binding, results in aggregation of the membrane-bound IgE and causes immediate mast cell degranulation and release of inflammatory mediators. Mast cells possess in their membranes IgE monomers able to recognize different antigenic epitopes. This contrasts with each single B lymphocyte, which possesses IgM antibody on its surface membrane that performs a cognitive function but is capable of recognizing only a single epitope specificity.
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3.4.7 Neonatal immunity The neonate lacks the ability to mount a full immunological response, accordingly maternal IgG is transported across the placenta late in pregnancy and is also absorbed across the gastrointestinal tract from breast milk. Maternal IgA secreted into breast milk will also provide mucosal protection for the neonate. 3.4.8 Activation of the classical complement pathway A complement cascade similar to that of the alternative pathway can be activated through specific antibody–antigen interactions. The antibodies that activate the classical complement pathway are IgM and IgG. Key steps in the activation of the classical pathway are shown in Fig. 8.7, where a contrast is also drawn to the alternative pathway. In the classical pathway the initiating step is the specific binding of IgG or IgM to antigen. Once this occurs, a complement protein termed C1 (which comprises a single C1q subunit, two C1r subunits and two C1s subunits) binds to adjacent Fc domains in the antibody–antigen complex. This binding of C1 activates the catalytic activity of the C1r subunits, and in turn the C1s subunits. The activated C1s subunits cleave C4 into C4b and C4a; the latter can diffuse away and serve as a leucocyte activator. The C4b covalently associates with the antibody –antigen complex on the surface of a microbial membrane and can serve as an opsonin. A further complement protein, C2, binds to this membrane complex to give C4b2. The C1s subunit then enzymically cleaves the bound C2a to generate on the membrane a new complex termed C4b2b, which is the C3 convertase of the classical pathway. (In some texts the C2a is referred to as the larger subunit remaining with the membrane while C2b is the smaller subunit that diffuses away.) This C3 convertase molecule is distinct from that within the alternative pathway, but it is from this point onwards that parallels can be drawn between the two cascades. The host proteins that serve key regulatory functions within the alternative pathway (DAF, CR1
Basic aspects of the structure and functioning of the immune system
Classical
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Fig. 8.7 The classical and alternative
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complement pathways.
factor I, CD59) also serve similar functions within the classical pathway. However, in contrast to the alternative pathway the activation step in the classical pathway requires specific antibody–antigen interactions. In this context the C1 protein can only become catalytically active when it is bound to at least two adjacent Fc domains. In the case of the IgG and IgM molecules the Fc domains will only align adjacent to each other when the corresponding Fab domains bind antigen. Further, when C1 is free in the circulation it is bound to a protein termed C1 inhibitor (C1-INH) which prevents any possible activation of C1 in the absence of antibody. Once C1 binds to adjacent Fc domains within an antibody–antigen complex C1-INH is displaced. The functions of the classical complement pathway are similar to those described for the alternative pathway, i.e. opsonization, leucocyte activation and membrane lysis of target cells. The classical pathway can additionally lead to complement protein deposition upon insoluble antibody–antigen immune complexes circulating within blood, and in doing so promote the clearance of such potentially harmful complexes by Kupffer cells of the liver. The presence of two complement pathways provides for rapid (alternative) and specific (classical) activation of a key defence mechanism, and offers greater protection
against the development of microbial resistance mechanisms.
4 Cell-mediated adaptive immune system Cell-mediated adaptive immune responses are mediated by T lymphocytes which arise from bone marrow progenitor cells and undergo maturation in the thymus before release into the systemic blood and lymph circulations (Fig. 8.1). There are a number of parallels that can be drawn between the B-lymphocyte-mediated immune response and the T-lymphocyte-mediated response. First, membrane-bound antibodies serve the cognitive function for B lymphocytes, while the cognitive function for T lymphocytes is served by T-cell receptors (TCR) present on the cell’s plasma membrane surface. Second, in response to antigen T lymphocytes, like B lymphocytes, will undergo clonal proliferation and form a population clone of memory T cells specific for a single epitope. Third, each single B lymphocyte and plasma cell that derives from it is capable of producing antibodies that will only recognize a single epitope. In the same manner each single T cell is programmed to make T-cell receptors of only a single specificity able to recognize only a single specific set of chemical features within a T131
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cell epitope. This is achieved for the TCR in a similar manner to that for antibodies in that different gene segments termed V-D-J are brought together by the process of gene rearrangement into single RNA products. The recombined RNA will code for the polypeptide chains that make up the TCR. When all the possible recombinations are considered, the number of different TCR molecules that an individual can make is in excess of 109, a number similar to the primary antigen recognition repertoire of T cells. There are two general classes of T lymphocytes: helper T lymphocytes and cytotoxic T lymphocytes. The latter function to kill host cells that have undergone a transformation such as a viral infection or cancer and they recognize specific antigens on the surface of host cells that have arisen as a result of such cell transformation. Through this specific antigen recognition, the cytotoxic T cell becomes closely apposed to the target host cell and is activated to synthesize and release cytotoxic secretory products (e.g. pore-forming molecules such as perforins) leading to lysis of the affected host cell. In contrast, the helper T cell can be viewed as the coordinator of the adaptive immune system, providing appropriate activation signals, in the form of secreted cytokines, to promote the functioning of both the cytotoxic T-cell populations and that of the antibody-producing B-lymphocyte and plasma cell populations. The actions of the helper T-cell populations also promote the function of the innate immune system, for example IFN-g released by helper T cells increases the phagocytic activity of macrophages. The helper T cells are further divided into Th1 and Th2 subpopulations depending upon the nature of cytokines they secrete and, as a consequence, the arms of the immune system they predominantly influence. The Th1 helper T cells mainly regulate cell-mediated immunity through expression of cytokines such as IL-2, IL-12 and IFN-g, which modulate cytotoxic T-cell and NK cell function. The Th2 helper T cells regulate humoral immunity through expression of cytokines such as IL-2, IL-4, IL-5, IL-6 and IL-10.
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4.1 T-lymphocyte antigen recognition and MHC proteins Epitopes for T lymphocytes comprise exclusively linear peptide sequences. T lymphocytes are unable to respond to carbohydrate, lipid or nucleic acid material and they only respond to peptide antigen when it is presented to the T lymphocyte by surface proteins on the plasma membrane of host cells. These surface proteins are termed major histocompatibility complex (MHC) proteins and can be subdivided into two main classes. MHC class I proteins are expressed on the surface of all nucleated host cell membranes and present peptide antigen to cytotoxic T lymphocytes. MHC class II proteins are expressed only on a more specialized group of cells termed antigen-presenting cells (APCs), and present peptide antigen to helper T lymphocytes. Such a distinct cellular distribution of MHC proteins and restriction in presentation to discrete T-lymphocyte subpopulations may be remembered by considering the different T-lymphocyte functions. That is, all cells of the body have the potential to become infected with virus and undergo a cancerous change and hence all cells must have the capacity to be destroyed by the actions of cytotoxic T cells. As such, all cells of the body must possess MHC I molecules to afford antigen presentation to cytotoxic T cells. In contrast, as a co-ordinator cell of the immune system, the helper T cell must be able to respond to its environment in order to give appropriate signals or ‘help’ to other immune cells. Specialized APCs with the capacity to phagocytose interstitial proteinaceous material therefore undertake the function of ‘environmental sampling’. MHC class II proteins expressed on the surface of APCs will present peptide antigen to helper T lymphocytes. APCs include the macrophage tissue cell population, specialized APCs such as dendritic cells within the lymphatic system or Langerhans cells within the skin. B lymphocytes also serve the function of an APC because they interact with protein antigen through high affinity surface IgM molecules. Subsequently, they internalize the protein antigen for processing to generate peptides that will be presented by MHC II molecules expressed on the B-
Basic aspects of the structure and functioning of the immune system
(a)
(b)
Cytotoxic (CD8+) T-cell
Helper (CD4+) T-cell
CD3
CD3
T-cell membrane
T-cell membrane TCR
Fig. 8.8 The process of MHC
presentation of peptide and interaction with T-lymphocyte receptor (TCR). Cytotoxic T lymphocytes (CD8+) interact with MHC I (a), while helper T lymphocytes (CD4+) interact with MHC II (b).
CD8
MHC I
TCR CD4
MHC II APC membrane
Host cell membrane
lymphocyte cell surface. Another cell type that can serve the function of an APC is the endothelial cell, which can be induced to express MHC II molecules by the action of the cytokine IFN-g. It should not be overlooked that the APC can itself become infected with virus and undergo cancerous transformation, and therefore the APC, in addition to MHC II molecules, will also express the full complement of MHC I molecules on its surface. This process of MHC presentation of peptide and interaction with T-lymphocyte receptor is shown in Fig. 8.8. A foreign peptide presented by a MHC molecule will be recognized by a TCR expressed on the surface of an appropriate T lymphocyte. Once a particular TCR recognizes a peptide sequence as foreign, intracellular signals to activate the T cell are sent via the CD3 complex present within the T-cell membrane. The recognition of peptide as foreign will lead to an immune response. Beyond antigen presentation, the interaction between MHC molecule and T lymphocyte also serves to identify that the T lymphocyte and host cell membrane arise from the same embryonic tissue. Tremendous inter-individual differences or more specifically polymorphisms exist in the MHC proteins within a population. The T lymphocyte undertakes this MHC surveillance through the possession of accessory molecules. Cytotoxic T lymphocytes possess CD8+ molecules which interact with MHC I (Fig. 8.8a), while helper T lymphocytes possess CD4+ molecules which interact with MHC II (Fig. 8.8b); hence the use of the terms
Any host cell
APC
CD8+ lymphocytes to refer to cytotoxic T cells and CD4+ lymphocytes to refer to helper T cells. 4.2 Processing of proteins to allow peptide presentation by MHC molecules Peptide epitope presented by MHC I is derived from the processing of proteins (e.g. a viral protein) synthesized within the actual cell that eventually will present the peptide to cytotoxic T lymphocytes. The MHC I molecule is composed of two polypeptide chains, an a-chain which has a1, a2 and a3 domains, and a second polypeptide termed b2microglobulin. The a1 and b2 domains form a peptide-binding cleft which can accommodate peptides up to 11 amino acids in length. Figure 8.9a shows the processing of a protein into peptide fragments for presentation by MHC I. The synthesized protein (indicated by an asterisk) is present in the cytoplasm of the cell and is degraded by a subcellular organelle termed a proteasome. The derived peptide fragments are actively transported, via a TAP peptide transporter, into the lumen of the endoplasmic reticulum (ER) where they fit within the binding clefts of MHC I molecules. From the ER the MHC I with bound peptide is transported to the transGolgi network (TGN), from which it is transported via endosomes to the plasma membrane where the MHC I molecule with bound peptide is accessible to surveillance by cytotoxic CD8+ T lymphocytes. Peptide epitopes presented by MHC II are derived from proteins present within the extracellular 133
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(a)
(b) CYTOTOXIC (CD8+) T CELL
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MHC I MHC II
Protein * Endosome
Protein * Proteasome Endosome
Endosome
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TGN ER ER TAP peptide transporter MHC I
ANY HOST CELL
ANTIGEN-PRESENTING CELL
Fig. 8.9 Schematic of the processing of a protein into peptide fragments for presentation by MHC. (a) The synthesized protein (*) is present in the cytoplasm of the cell and is degraded into peptide by the proteasome. The derived peptide fragments are presented by MHC I to cytotoxic CD8+ T lymphocytes. (b) Protein (*) is endocytosed by an APC (antigen-presenting cell) and processed into peptide within lysosomes. The derived peptide fragments are presented by MHC II to helper CD4+ T lymphocytes.
fluid and are presented to helper T lymphocytes by APCs. The MHC II molecule is composed of two polypeptide chains, an a-chain which has a1 and a2 domains, and a b-chain which has b1 and b2 domains. The a1 and b1 domains form a peptidebinding cleft that can accommodate peptides up to 20 amino acids in length. Figure 8.9b shows the processing of a protein into peptide fragments for presentation by MHC II. In this case the protein (indicated by an asterisk) is internalized from the extracellular fluid by the APC and restricted to an endosomal compartment without access to the APC’s cytoplasm. The endosome delivers the protein to a lysosome compartment which degrades the protein into peptide fragments, after which the 134
peptide fragments are returned to an endosome compartment. In the lumen of the ER the MHC II molecule becomes associated with another protein termed an invariant chain which blocks access of peptides to the binding cleft of the MHC II molecule; The MHC II–invariant chain complex is transferred to the TGN and then to an endosomal compartment. At this point the endosomes that contain the processed peptide and the MHC II molecules merge, and the invariant chain disintegrates, allowing peptides access to the MHC II-binding cleft. The MHC II molecules with bound peptide are transported to the plasma membrane where they are accessible to surveillance by helper CD4+ T lymphocytes.
Basic aspects of the structure and functioning of the immune system
5 Some clinical perspectives This section is intended to provide a brief overview of some clinical issues that exemplify the basic aspects of immune system functioning discussed previously. 5.1 Transplantation rejection Transplantation is the process of transferring cells, tissues or organs — termed a graft — from one location to another. An autologous graft refers to a transplant between two sites within the same individual, e.g. skin graft from the thigh to the hand. An allogeneic graft refers to a transplant between two genetically different individuals of the same species, e.g. kidney transplant from a donor to a recipient individual. A xenogenic graft refers to a transplant across different species, e.g. pig to human. The tempo of clinical rejection, in for example kidney transplantation, is often categorized by the following stages: • Hyperacute rejection occurs within minutes to hours following revascularization of a graft. The cause is due to the presence of preformed circulating antibody (IgG) that reacts with the blood cell antigens (the ABO system), or MHC I molecules or other poorly defined antigens. This should now be a rare event clinically as recipients are tested (crossmatched) before transplantation for the presence of antibodies reactive with cells of the donor. • Acute rejection occurs within weeks to months following transplantation and involves humoral (antibody) and cell-mediated induced cytotoxicity. Damage may be reversed with early diagnosis and more aggressive immunosuppressive therapy. • Chronic rejection occurs many months to even years following transplantation. The pathology is characterized by fibrosis and may require differential diagnosis to distinguish between a chronic rejection event and the recurrence of the original disease that necessitated transplantation in the first place. The major alloantigens (i.e. antigens responsible for rejection of allogeneic grafts) are the MHC proteins. Although there are two distinct classes of MHC protein (described in section 4.1) the MHC molecules actually comprise a number of subclasses
which vary further in the general nature of peptides that they will accept within their binding clefts. The MHC I molecules are composed of three subclasses, MHC IA, MHC IB and MHC IC, on each nucleated cell of the body; all three subclasses are simultaneously expressed. The MHC II molecules are also composed of three subclasses, MHC II DR, MHC II DP and MHC II DQ, and again on each APC all three subclasses of MHC II molecule are simultaneously expressed. APCs, like other cells in the body, will also express MHC I molecules on their surface in addition to MHC II. As indicated previously, the major cause of allogeneic tissue transplantation rejection is the polymorphic nature of the MHC phenotype between individuals. Polymorphism in MHC arises within the population because the genes for each of the MHC subclasses can exist in multiple different forms or alleles. For example, in humans there are at least 52 different forms of the MHC IB gene and at least 24 different forms of the MHC IA gene. It follows that individuals in a population can possess any one of the 52 different forms of MHC IB gene and any one of the 24 different forms of MHC 1A gene, so the number of different combinations for the six classes of MHC proteins is many millions. The situation is further complicated by the fact that each individual inherits and co-expresses a set of MHC I and II genes from each parent. This means that on each nucleated cell of the body there will be coexpressed paternally derived and maternally derived versions of the MHC IA, MHC IB and MHC IC molecules. The same principle will apply for coexpression upon APCs of paternal and maternal MHC II protein subclasses. This tremendous polymorphism is important in immune defence because it allows the broadest possible scope of peptide antigen presentation, and thus the best chance of survival of a population as a whole, but it also confers the very high probability of MHC mismatch during allogeneic transplantation. As a result of the mode of MHC inheritance, the highest probability of a MHC tissue match between individuals that are not genetically identical twins will be that obtained between siblings, where there is a 1 in 4 chance of a sibling possessing an exact match for all the MHC I and MHC II subclasses. The MHC proteins are also termed human 135
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leucocyte antigens (HLA), and HLA tissue typing is undertaken routinely before transplantation to gain improved matches between donor and recipient. In kidney transplantation it has been found that matching the MHC IA, IB and IIDR genes in particular appears to improve short- and long-term graft survival. The main target for the modern immunosuppressants such as cyclosporin and tacrolimus is inhibition of cytokine gene transcription in a highly selective manner in the helper T-lymphocyte populations. The consequence of this is to inhibit helper T-cell auto-activation and helper T-cell co-activation of cytotoxic T lymphocytes and of B lymphocytes, and thus considerably ‘damp down’ cell-mediated and humoral immune responses to the graft. 5.2 Hypersensitivity Hypersensitivity can be defined as an exaggerated response of the immune system leading to host tissue damage. However, some of the immune responses described in the hypersensitivity classification below are, in some circumstances, appropriate responses to invading antigen. For example, a component in what is an appropriate immune response to tissue transplant rejection can be defined as a type II hypersensitivity reaction. On the basis of the highly influential Gell and Coombs’ classification scheme, there are four categories of hypersensitivity. • Type I — immediate hypersensitivity. This is also called anaphylactic or acute hypersensitivity. It involves IgE antibody and is mediated via degranulation of mast cells leading to release of preformed factors which promote an influx of immune cells to the site of mast cell activation and initiation of a rapid inflammatory reaction. In the extreme case the inflammatory response extends beyond the localized site of initiation and affects systemic tissues leading to life-threatening anaphylactic reactions such as those documented to penicillin, to peanut antigen, or to bee sting antigen. Examples of localized type I hypersensitivity would include hay fever. The term ‘allergy’ has become synonymous with type I hypersensitivity. • Type II hypersensitivity — antibody-mediated 136
cytotoxicity. This is caused by antibodies that are directed against cell surface antigens. IgG and IgM are the key antibodies involved that direct cytotoxic events against the cell surface with which they interact. The cytotoxic events would include activation of the classical complement pathway leading to the formation of a MAC, and the attraction and activation of killing cells such as NK cells or phagocytes which can bind to the antigen– antibody complex via receptors for antibody Fc domains or complement C3b. Type II hypersensitivity disorders include blood transfusion reactions arising from mismatch of the blood ABO antigens between donor and recipient, or haemolytic disease of the newborn. Autoimmune disorders such as myasthenia gravis, Goodpasture’s syndrome and autoimmune haemolytic anaemias are initiated by autoantibodies reacting against ‘self’ tissue. • Type III hypersensitivity — complex-mediated. This involves the formation of large antigen– antibody complexes that circulate in the blood, are usually coated by complement proteins and removed by phagocytosis. If this process is compromised for any reason then the antigen–antibody complexes will be deposited in tissue capillary beds, with kidney deposition being clinically the most important site. This deposition of high molecular weight antigen–antibody complexes in the glomerular capillaries of the kidney can lead to a condition termed glomerulonephritis which involves disruption of the glomerular basement membrane, destruction of glomeruli and ultimately renal failure which may necessitate organ transplantation. Systemic lupus erythematosus is a condition where autoantibodies are directed against the host’s DNA and RNA with subsequent complement-coated immune complexes deposited throughout systemic tissues such as in the kidney, skin, joints and brain. • Type IV hypersensitivity — cell-mediated. This results from inappropriate accumulation of macrophages at a localized site, and may or may not involve the presence of antigen. Under conditions of ongoing localized infection or inflammation, macrophages release proteases, which destroy infected or otherwise damaged tissue. However, with the inappropriate recruitment and/or activation of excessive numbers of macrophages, continuing
Basic aspects of the structure and functioning of the immune system
damage to normal tissue may result, leading to chronic inflammation. The recruitment and activation of macrophages in type IV hypersensitivity is augmented by the activity of helper T lymphocytes (specifically the Th1 subpopulation). Examples of type IV hypersensitivity include granuloma formation and contact dermatitis. Granulomas are initiated and maintained by the recruitment of macrophages into the site of a persistent source of antigen or toxic material. A granuloma is a fibrotic core of tissue composed of tissue cells and macrophages surrounded by lymphocytes and then further surrounded by layers of calcified collagenous material. The disease sarcoidosis is a granulomatous disease of unknown cause but characterized by granuloma nodule formation in the lung and skin, among other sites.
6 Summary The immune system is a complex body system whose various functions display a high level of inter-regulation. As such, any attempt to describe the functioning of the immune system within a single chapter will inevitably represent an oversimplification. However, the authors consider this chapter to be a comprehensive, but nevertheless basic, overview of the immune system that will serve as a sound foundation for further reading on the clinical immunological basis of disease or for the consultation of more specialized texts on immunological function. The discussion in this chapter is structured by delineating the immune system into innate and
adaptive responses. The innate system, responding immediately but non-specifically to antigen, is complimentary to the adaptive immune system which reacts in a highly specific manner to antigen but which displays a delay in its response. It should not be forgotten, however, that the functionings of the two systems are intimately related, showing dependency upon each other for the optimal maintenance of health.
7 Further reading Abbas, A. K., Lichtman, A. H. & Pober, J. S. (2000) Cellular and Molecular Immunology. WB Saunders, Philadelphia and London. [Strong on experimental observations that form the basis for the science of immunology at the molecular, cellular, and whole-organism levels, and the resulting conclusions.] Clancy, J. & Morgan, J. (2001) Basic Concepts in Immunology: A Student’s Survival Guide. McGraw-Hill, New York and London. [Foundation text.] Janeway, C., Travers, P., Capra, J. D. & Walport, M. J. (2001) Immunobiology: The Immune System in Health and Disease. Garland Press, New York. [Medical and basic immunology with emphasis on concepts.] Parkin, J. & Cohen, B. (2001) An overview of the immune system. Lancet, 357, 1777–1789. Playfair, J. H. L. (2000) Immunology at a Glance. Blackwell Science, Oxford. [Pictorial based primer for immunological novices.] Roitt, R. & Delves, P. J. (2001) Roitt’s Essential Immunology, 10th edn. Blackwell Science, Oxford. [Classic introductory text.] Roitt, I. & Rabson, A. (2000) Really Essential Medical Immunology. Blackwell Science, Oxford. [Contains essential immunological information for medical students and other health professionals.]
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Chapter 9
Vaccination and immunization Peter Gilbert and David Allison 1 Introduction 2 Spread of infection 2.1 Common source infections 2.2 Propagated source infections 3 Objectives of a vaccine/immunization programme 3.1 Severity of the disease 3.2 Effectiveness of the vaccine/immunogen 3.3 Safety 3.4 Cost 3.5 Longevity of the immunity 4 Classes of immunity 4.1 Passive acquired immunity 4.2 Active acquired immunity 5 Classes of vaccine 5.1 Live vaccines 5.2 Killed and component vaccines 5.3 DNA vaccines 6 Routine immunization against infectious disease
6.1 Poliomyelitis vaccination 6.2 Measles, mumps and rubella vaccination (MMR) 6.2.1 Measles 6.2.2 Mumps 6.2.3 Rubella 6.2.4 MMR vaccine 6.3 Tuberculosis 6.4 Diphtheria, tetanus and pertussis (DTP) immunization 6.4.1 Diphtheria 6.4.2 Tetanus 6.4.3 Pertussis (whooping cough) 6.4.4 DTP vaccine combinations and administration 6.5 Haemophilus influenzae type b (Hib) immunization 6.6 Meningococcal immunization 7 Juvenile immunization schedule 8 lmmunization of special risk groups 9 Further reading
1 Introduction
smallpox lesions. Such treatments produced single localized lesions and commonly, but not always, protected the recipient from contracting full-blown smallpox. The process became known as variolation and, unknown to its practitioners, attenuated the disease through changing the route of infection of the causal organism from respiratory transmission to cutaneous. Unfortunately, occasional cases of smallpox resulted from such treatment. Further developments recognized that immunity developed towards one disease often brings with it crossimmunity towards another related condition. Cowpox is a disease of cattle that can be transmitted to man. Symptoms are similar but less severe than those of smallpox. Material taken from active cowpox (vaccinia) lesions was therefore substituted into the variolation procedures. This conferred much of the protection against smallpox that had become associated with variolation but without the associated risks. This discovery, by Edward Jenner, made over two centuries ago, became known as
People rarely suffer from the same infectious disease twice. When such re-infections occur it is with an antigenically modified strain (common cold, influenza), the patient is immunocompromised (immunosuppressive drugs, immunological disorders) or a long time has elapsed since the first infection. Alternatively the patient may have failed to eliminate the primary infection that has then remained latent and emerges in a modified or similar form (herpes simplex, cold sores; herpes zoster, chickenpox). Immunity against re-infection was recognized long before the discovery of the causal agents of infectious disease. Efforts were therefore made towards developing treatment strategies that might generate immunity to infection. An early development was the attempted control of smallpox (variola major) through the deliberate introduction, under the skin of healthy individuals, of material taken from active 138
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Fig. 9.1 Reported incidence of
paralytic poliomyelitis in England and Wales during the 1950s and 1960s. After the introduction of vaccination programmes the incidence of this disease dropped from an endemic incidence of c. 5000 cases per year to fewer than 10.
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vaccination and heralded a new era in disease control. The term vaccination is now widely used to describe prophylactic measures that use living microorganisms or their products to induce immunity. The more general term immunization describes procedures that induce immunity in the recipient but which do not necessarily involve the use of microorganisms. Nowadays vaccination and immunization procedures are used not only to protect the individual against infection but also to protect communities against epidemic disease. Such public health measures have met with spectacular success, as illustrated in Fig. 9.1 for the incidence of paralytic poliomyelitis. In those instances where there is no reservoir of the pathogen other than in infected individuals, and survival outside the host is limited (i.e. smallpox, poliomyelitis and measles) then such programmes, worldwide, have the potential to eradicate the disease permanently.
2 Spread of infection Infectious diseases may either be spread from a
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common reservoir of the infectious agent that is distinct from the diseased individuals (common source) or they might transfer directly from a diseased individual to a healthy one (propagated source). 2.1 Common source infections In common source infections, the reservoir of infection might be animate (e.g. insect vectors of malaria and yellow fever) or they might be inanimate (infected drinking water, cooling towers, contaminated food supply). In the simplest of cases the source of the infection is transient (i.e. food sourced to a single retail outlet, or to an isolated event such as a wedding reception). In such instances the onset of new cases is rapid, phased over 1–1.5 incubation periods, and the decline in new cases closely follows the elimination of the source (Fig. 9.2). This leads to an acute outbreak of infection limited socially and geographically to those linked with the source. Such an incident was epitomized by the outbreak of Escherichia coli O157 infections in Lanarkshire in the winter of 1996 that was linked to a single retail 139
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outlet. If the source of the infection persists beyond the onset, then the incidence of new cases is maintained at a level that is commensurate with the infectivity of the pathogen and the frequency of exposure of individuals. In this manner, if cases of the variant Creutzfeldt–Jakob disease (vCJD), first recognized in the mid-1990s, relate to human exposure to bovine spongiform encephalopathy (BSE)infected beef in the early 1980s, then the incidence of vCJD will increase over 1–1.5 incubation periods (i.e. 10–15 years) and be sustained for as long as such meat had remained on sale. In such a scenario vCJD related to a single common source outbreak, could persist for another 20–30 years. For those infectious diseases that are transmitted to humans via insect vectors then onset and decline phases of epidemics are rarely observed other than as reflections of the seasonal variation in the prevalence of the insect. Rather, the disease is endemic within the population group and has a steady incidence of new cases. Diseases such as these are generally controlled by public health measures and environmental control of the vector with vaccination and immunization being deployed to protect individuals (e.g. yellow fever vaccination). 140
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outbreaks of infection where the source persists (䊏) and where it is short-lived (䊉).
2.2 Propagated source infections Propagated outbreaks of infection relate to the direct transmission of an infective agent from a diseased individual to a healthy, susceptible one. Mechanisms of such transmission were described in Chapter 7 and include inhalation of infective aerosols (measles, mumps, diphtheria), direct physical contact (syphilis, herpes virus) and, where sanitation standards are poor, through the introduction of infected faecal material into drinking water (cholera, typhoid) or onto food (Salmonella, Campylobacter). The ease of transmission, and hence the rate of onset of an epidemic (Fig. 9.3) relates not only to the susceptibility status and general state of health of the individuals concerned but also to the virulence properties of the organism, the route of transmission, the duration of the infective period associated with the disease, behavioural patterns, age of the population group and the population density (i.e. urban versus rural). Each infective individual will be capable of transmitting the disease to the susceptible individuals that they encounter during their infective period. The number of persons to which a single infective
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Fig. 9.3 Propagated outbreaks of infection
showing the incidence of new cases (䊉), diseased individuals (䊏) and recovered immune (䉱). The dotted line indicates the incidence pattern for an incompletely mixed population group.
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individual might transmit the disease, and hence the rate of occurrence of the infection within the population, will depend upon the population density with respect to susceptible and infective individuals, the degree and nature of their social interaction, and the duration and timing of the infective period. Clearly if infectivity precedes the manifestation of disease, then spread of the infection will be greater than if these were concurrent. As each infected individual will, in turn, become a source of infection then this leads to a near exponential increase in the incidence of disease. Figure 9.3 shows the incidence of disease within a theoretical population group. This hypothetical group is perfectly mixed, and all individuals are susceptible to the infection. The model infection has an incubation period of 1 day and an infective period of 2 days commencing at the onset of symptoms with recovery occurring 1 day later. For the sake of this illustration it has been assumed that each infective individual will infect two others per day until all of the population group have contracted the disease (solid lines). In practice, however, the rate of transmission will decrease as the epidemic progresses, as the recovered individuals will become immune to further infection, reducing
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the population density of susceptible individuals, and thereby the likelihood of onward transmission. Epidemics therefore often cease before all members of the community have been infected (Fig. 9.3, dotted line). If the proportion of immune individuals within a population group can be maintained above this threshold level then the likelihood of an epidemic arising from a single isolated infection incident is small (herd immunity). The threshold level itself is a function of the infectivity of the agent and the population density. Outbreaks of measles and chickenpox therefore tend to occur annually in the late summer among children attending school for the first time. This has the effect of concentrating all susceptible individuals in one, often confined, space and thereby reducing the proportion of immune subjects to below the threshold for propagated transmission. An effective vaccination programme is therefore one that can maintain the proportion of individuals who are immune to a given infectious disease above the critical threshold level. Such a programme will not prevent isolated cases of infection but will prevent these from becoming epidemic.
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3 Objectives of a vaccine/immunization programme There is the potential to develop a protective vaccine/immunization programme for each and every infectious disease. Whether or not such vaccines are developed and deployed is related to the severity and economic impact of the disease upon the community as well as the effects upon the individual. Various factors governing the likelihood of an immunization programme being adopted are discussed below, while the principles of immunity, and of the production and quality control of immunological products are discussed in Chapters 8 and 23, respectively. 3.1 Severity of the disease The severity of the disease, not only in terms of its morbidity and mortality and the probability of permanent injury to its survivors, but also in the likelihood of infection must be sufficient to warrant the development and routine deployment of a vaccine and its subsequent use. Thus, whilst influenza vaccines are constantly reviewed and stocks maintained, the control of influenza epidemics through vaccination is not recommended. Rather, those groups of individuals, such as the elderly, who are at special risk from the infection are protected. Vaccines to be included within a national immunization and vaccination programme are chosen to reflect the infection risks within that country. Additional immunization, appropriate for persons travelling abroad, is intended not only to protect the at-risk individual, but also to prevent importing the disease into an unprotected home community. 3.2 Effectiveness of the vaccine/immunogen Vaccination and immunization programmes seldom confer 100% protection against the target disease. More commonly the degree of protection is c. 60–95%. In such instances, while individuals receiving treatment will have a high probability of becoming immune, virtually all members of a community must be treated in order to reduce the actual proportion of susceptible individuals to below the threshold for epidemic spread of the dis142
ease. Anti-diphtheria and anti-tetanus prophylaxis, which utilize toxoids, are among the most efficient immunization programmes, whereas the performance of BCG is highly variable, and cholera vaccine (killed) gives little personal protection and is virtually useless in combating epidemics, 3.3 Safety No medical or therapeutic procedure comes without some risk to the patient. All possible steps are taken to ensure safety, quality and efficacy of vaccines and immunological products (Chapter 23). The risks associated with immunization procedures must be constantly reviewed and balanced against the risks of, and associated with, contracting the disease. In this respect, smallpox vaccination in the UK was abandoned in the mid-1970s as the risks associated with vaccination then exceeded the predicted number of deaths that would follow importation of the disease. Shortly after this, in May 1980, the World Health Assembly pronounced the world to be free of smallpox. Similarly, the incidence of paralytic poliomyelitis in the USA and UK in the late 1990s was low with the majority of cases being related to vaccine use. As the worldwide elimination of poliomyelitis approaches, there is much debate as to the value of the live (Sabin) vaccine outside of an endemic area. Public confidence in the safety of vaccines and immunization procedures is essential if compliance is to match the needs the community. In this respect public concern and anxiety, in the mid-1970s, over the perceived safety of pertussis vaccine led to a reduction in coverage of the target group from c. 80% to c. 30%. Major epidemics of whooping cough, with over 100 000 notified cases, followed in 1977–1979 and 1981–1983. By 1992, public confidence had returned, coverage had increased to 92% and there were only 4091 reported cases. Similarly, links have been claimed between the incidence of autism in children and the change in the UK from single measles and German measles vaccines to the combined MMR vaccine. Such claims are unfounded but have nevertheless decreased the uptake of the MMR vaccine and, thereby, increased the likelihood and magnitude of future measles epidemics.
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3.4 Cost Cheap effective vaccines are an essential component of the global battle against infectious disease. It was estimated that the 1996 costs of the USA childhood vaccination programme, directed against polio, diphtheria, pertussis, tetanus, measles and tuberculosis, was $1 for the vaccines and $14 for the programme costs. The newer vaccines, particularly those that have been genetically engineered, are considerably more expensive, putting the costs beyond the budgets of many developing countries. 3.5 Longevity of the immunity The ideal of any vaccine is to provide lifelong protection of the individual against disease. Immunological memory (Chapter 8) depends on the survival of cloned populations of small B and T lymphocytes (memory cells). These small lymphocytes have a lifespan in the body of c. 15–20 years. Thus, if the immune system is not boosted, either by natural exposure to the organism or by re-immunization, then immunity gained in childhood will be attenuated or lost completely by the age of 30. Those vaccines that provide only poor protection against disease have proportionately reduced time-spans of effectiveness. Equally many of the immunization protocols are less effective, and have less duration, when administered to immunologically naïve individuals (neonates) rather than adults. Yellow fever vaccination, which is highly effective, must therefore be repeated at 10-year intervals, while typhoid vaccines are only effective for 1–3 years. Whether or not immunization in childhood is boosted at adolescence or in adult life depends on the relative risks associated with the infection as a function of age.
or accidental exposure to microorganisms or their component parts. Active acquired immunity might involve either or both of the humoral and cellmediated responses. 4.1 Passive acquired immunity Humoral antibodies of the IgG class are able to cross the placenta from mother to fetus. These antibodies will provide passive protection of the newborn against those diseases which involve humoral immunity and to which the mother is immune. In this fashion, newborn infants in the UK have passive protection against tetanus, but not against tuberculosis. The latter requires cell-mediated immunity. Secreted antibodies are also passed to the gut of newborn together with the first deliveries of breast milk (colostrum). Such antibodies provide some passive protection against infections of the gastrointestinal tract. Maternally acquired antibodies will react not only with antigen associated with a threatening infection but also with antigens introduced to the body as part of an immunization programme. Premature immunization, i.e. before degradation and elimination of the maternal antibodies, will therefore reduce the potency of an administered vaccine. This aspect of the timing of a course of vaccinations is discussed later. Administration of preformed antibodies, taken from animals, from pooled human serum, or from human cell lines is often used to treat an existing infection (e.g. tetanus, diphtheria) or condition (venomous snake bite). Pooled human serum may also be administered prophylactically, within a slowrelease vehicle, for those persons entering parts of the world where diseases such as hepatitis A are endemic. Such administrations confer no long-term immunity and will interfere with concurrent vaccination procedures.
4 Classes of immunity The theoretical background that underlies immunity to infection has been discussed in detail in Chapter 8. Immunity to infection may be passively acquired through the receipt of preformed, protective antibodies or it may be actively acquired through an immune response following deliberate
4.2 Active acquired immunity Active acquired immunity (Chapter 8) relates to exposure of the immune system to antigenic materials. Such exposure might be related to a naturally occurring or vaccine-associated infection, or it might be associated with the direct introduction of non143
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viable antigenic material into the body. The latter might occur through insect or animal bites and stings, inhalation, ingestion or deliberate injection. The route of exposure to antigen will influence the nature of the subsequent immune response. Thus, injection of antigen will lead primarily to humoral (IgG, IgM) production, while exposure of epithelial tissues (gut, respiratory tract) will lead not only to the production of secretory antibodies (lgA, lgE) but also, through the common immune response, to a stimulation of humoral antibody. The magnitude and specificity of an immune response depends not only upon the duration of the exposure to antigen but also upon its timeconcentration profile. During a naturally occurring infection the levels of antigen in the host are very small at onset and localized to the portal of entry to the host. As the amounts of antigen are small they will react only with a small, highly defined subgroup of small lymphocytes. These will undergo transformation to produce various antibody classes specific to the antigen and undergo a clonal expansion of their number. These immune responses and the progress of the infection will progress simultaneously. With time microorganisms will release greater amounts of antigenic materials that will, in turn, react with an increasing number of cloned lymphocytes, to produce yet more antibody. Eventually the antibody levels will be sufficient to bring about the elimination, from the host, of the infecting organism. Antibody levels will then decline with the net result of this encounter being the clonal expansion of particular small lymphocytes relating to a highly specific ‘immunological memory’ of the encounter. This situation should be contrasted with the injection of a non-replicating immunogen. Often the amount of antigen introduced is large when compared with the levels present during the initial stages of an infection. In a non-immune animal the antigens will react not only with those lymphocytes that are capable of producing antibody of high specificity but also with those of a lower specificity. Antibody (high and low specificity) produced will react with and remove the residual antigen. The immune response will cease after this initial (primary) challenge. On a subsequent (secondary) challenge the antigen will react with residual preformed anti144
body relating to the first challenge together with a more specific subgroup of the original cloned lymphocytes. As the number of challenges is increased then the proportion of stimulated lymphocytes that are specific to the antigen becomes increased. After a sufficient number of consecutive challenges the magnitude and specificity of the immune response matches that which would occur during a natural infection with an organism bearing the antigen. This pattern of exposure brings with it certain problems. Firstly, as the introduced immunogen will react preferentially with preformed antibody rather than lymphocytes then sufficient time must elapse between exposures so as to allow the natural loss of antibody to occur. Secondly, immunity to infection will only be complete after the final challenge with immunogen. Thirdly, low specificity antibody produced during the early exposures to antigen might be capable of cross-reaction with host tissues to produce an adverse response to the vaccine.
5 Classes of vaccine Vaccines may be considered as representing live microorganisms, killed microorganisms or purified bacterial and viral components (component vaccines). Recent innovations have included the introduction of DNA vaccines that encode for the transcription of antigen when introduced directly into host tissues or that might be delivered by virus vectors (i.e. adenovirus). Some aspects of these vaccine classes are discussed below. 5.1 Live vaccines Vaccines may be live, infective microorganisms, attenuated with respect to their pathogenicity but retaining their ability to infect, or they might be genetically engineered such that one mildly infective organism has been modified so that it causes the expression of antigens from an unrelated pathogen. Two major advantages stem from the use of live vaccines. Firstly, the immunization mimics the course of a natural infection such that only a single exposure is required to render an individual immune. Secondly, the exposure may be mediated through the natural route of infection (e.g. oral)
Vaccination and immunization
thereby stimulating an immune response that is appropriate to a particular disease (e.g. secretory antibody as a primary defence against poliomyelitis virus in the gut). Disadvantages associated with the use of live vaccines are also apparent. Live attenuated vaccines, administered through the natural route of infection, will be replicated in the patient and could be transmitted to others. If attenuation is lost during this replicative process then infections might result (see poliomyelitis, below). A second, major disadvantage, of live vaccines is that the course of their action, and possible side-effects, might be affected by the infection and immunological status of the patient. 5.2 Killed and component vaccines As these vaccines are unable to evoke a natural infection profile with respect to the release of antigen then they must be administered on a number of occasions. Immunity is not complete until the course of immunization is complete and, with the exception of toxin-dominated diseases (diphtheria, tetanus) where the immunogen is a toxoid, will never match the performance of live vaccine delivery. Specificity of the immune response generated in the patient is initially low. This is particularly the case when the vaccine is composed of a relatively crude cocktail of killed cells where the immune response is directed only partly towards antigenic components of the cells that are associated with the infection process. This increases the possibility of adverse reactions in the patient. Release profiles of these immunogens can be improved through their formulation with adjuvants (Chapters 8 and 23), and the immunogenicity of certain purified bacterial components such as polysaccharides can be improved by their conjugation to a carrier. 5.3 DNA vaccines A recent development, associated with research into gene therapy, has been the use of DNA encoding specific virulence factors of defined pathogens to evoke an immune response. The DNA is introduced directly into tissue cells by means of a transdermal gene-gun and is transcribed by the recipient
cells. Accordingly the host responds to the antigenic material produced as though it were an infection. The course of release of the antigen reflects that of a natural infection and, therefore, a highly specific response is invoked. Eventually the introduced DNA is lost from the recipient cells and antigen release ceases. To date the approach has been used with some success in veterinary medicine but has not yet been employed for humans.
6 Routine immunization against infectious disease 6.1 Poliomyelitis vaccination Poliovirus, a picornavirus, has three immunologically distinct types (I, II and III). The first phase of poliomyelitis infection is an acute infection of lymphoid tissues associated with the gastrointestinal tract (Peyer’s patches), during which time the virus can be found in the throat and in faeces. The second phase is characterized by an invasion of the bloodstream, and in the third phase the virus migrates from the bloodstream into the meninges. Infections range in severity from clinically inapparent (>90%) to paralytic. Paralytic poliomyelitis is a major illness, but only occurs in 0.1–2% of infected individuals. It is characterized by the destruction of large nerve cells in the anterior horn of the brain resulting in varying degrees of paralysis. Unvaccinated adults are at greater risk from paralytic infection than children. The infection is transmitted by the faecal–oral route. Polio is the only disease, at present, for which both live and killed vaccines compete. Since the introduction of the killed virus (Salk) in 1956 and the live attenuated virus (Sabin) in 1962 there has been a remarkable decline in the incidence of poliomyelitis (Fig. 9.1). The inactivated polio vaccine (IPV) contains formalin-killed poliovirus of all three serotypes. On injection, the vaccine stimulates the production of antibodies of the IgM and IgG class that neutralize the virus in the second stage of infection. A course of three injections at monthly intervals produces long-lasting immunity to all three poliovirus types. The live, oral polio vaccine (OPV) is widely used 145
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in many countries, including the UK and USA. Its main advantages over the IPV vaccine are its lower cost and easier administration. OPV contains attenuated poliovirus of each of the three types and is administered, as a liquid, onto the tongue. The vaccine strains infect the gastrointestinal mucosa and oropharynx, promoting the common immune response, and involving both humoral and secretory antibodies. IgA, secreted within the gut epithelium, provides local resistance to the first stages of poliomyelitis infection. OPV therefore provides protection at an earlier stage of the infection than does IPV. Infection of epithelial cells with one strain of enterovirus, however, often inhibits simultaneous infection by related strains. At least three administrations of OPV are therefore required, with each dosing conferring immunity to one of the vaccine serotypes. These doses must be separated by a period of at least 1 month in order to allow the previous infection to lapse. Booster vaccinations are also provided to cover the eventuality that some other enterovirus infection, present at the time of vaccination, had reduced the response to the vaccine strains. Faecal excretion of vaccine virus will occur and may last for up to 6 weeks post-treatment. Such released virus will spread to close contacts and infect/(re-)immunize them. Vaccine-associated poliomyelitis may occur through reversion of the attenuated strains to the virulent wild-type, particularly with types II and III and is estimated to occur once per 4 million doses. As the wild-type virus can be isolated in faeces, infection may occur in unimmunized contacts as well as vaccine recipients. Since the introduction of OPV, notifications of paralytic poliomyelitis in the UK have dropped spectacularly. However, from 1985 to 1995, 19 of the 28 notified cases of paralytic poliomyelitis were associated with revertant vaccine strains (14 recipients, 5 contacts). As the risk of natural infections with poliomyelitis within developed countries has now diminished markedly, the greater risk resides with the live vaccine strains. Proposals are therefore now being considered that in the developed world OPV should be replaced with IPV.
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6.2 Measles, mumps and rubella vaccination (MMR) Measles, mumps and rubella (German measles) are infectious diseases, with respiratory routes of transmission and infection, caused by members of the paramyxovirus group. Each virus is immunologically distinct and has only one serotype. Whilst the primary multiplication sites of these viruses are within the respiratory tract, the diseases are associated with viral multiplication elsewhere in the host. 6.2.1 Measles Measles is a severe, highly contagious, acute infection that frequently occurs in epidemic form. After multiplication within the respiratory tract the virus is transported throughout the body, particularly to the skin where a characteristic maculopapular rash develops. Complications of the disease can occur, particularly in malnourished children, the most serious being measles encephalitis that can cause permanent neurological injury and death. A live vaccine strain of measles was introduced in the USA in 1962 and to the UK in 1968. A single injection produces high-level immunity in over 95% of recipients. Moreover, as the vaccine induces immunity more rapidly than the natural infection, it may be used to control the impact of measles outbreaks. The measles virus cannot survive outside of an infected host. Widespread use of the vaccine therefore has the potential, as with smallpox, of eliminating the disease worldwide. Mass immunization has reduced the incidence of measles to almost nil, although a 15-fold increase in the incidence was noted in the USA between 1989 and 1991 because of poor compliance with the vaccine. 6.2.2 Mumps Mumps virus infects the parotid glands to cause swelling and a general viraemia. Complications include pancreatitis, meningitis and orchitis, the latter occasionally leading to male sterility. Infections can also cause permanent unilateral deafness at any age. In the absence of vaccination, infection occurs in >90% of individuals by age 15 years. A live attenuated mumps vaccine has been available since 1967
Vaccination and immunization
and has been part of the juvenile vaccination programme in the UK since 1988 when it was included as part of the MMR triple vaccine (see below). 6.2.3 Rubella Rubella is a mild, often subclinical infection that is common among children aged between 4 and 9 years. Infection during the first trimester of pregnancy brings with it a major risk of abortion or congenital deformity in the fetus (congenital rubella syndrome, CRS). Rubella immunization was introduced to the UK in 1970 for prepubertal girls and non-immune women intending to start families. The vaccine utilizes a live, cold-adapted Wistar RA2713 vaccine strain of the virus. The major disadvantage of the vaccine is that, as with the wild-type, the fetus can be infected. While there have been no reports of CRS associated with use of the vaccine, the possible risk makes it imperative that women do not become pregnant within 1 month of vaccination. Prepubertal girls were immunized to extend the period of immunity through the child-bearing years. Until 1988 boys were not routinely protected against rubella. Their susceptibility to the virus was thought to maintain the natural prevalence of the disease in the community and thereby reinforce the vaccineinduced immunity in vaccinated, adult females. This proved to be not the case. Rather cases of CRS could be related to incidence of the disease in younger children within the family. Rubella vaccine is now given to both sexes at the age of 12–15 months as part of the MMR programme (below). 6.2.4 MMR vaccine MMR vaccine was introduced to the UK in 1988 for young children of both sexes, replacing single antigen measles vaccine. It consists of a single dose of a lyophilized preparation of live attenuated strains of the measles, mumps and rubella viruses. MMR had previously been deployed in the USA and Scandinavia for a significant number of years without any indication of increased adverse reaction or of decreased sero-conversion over separate administration of the component parts. Immunization results in sero-conversion to all three viruses in >95% of re-
cipients. For maximum effect MMR vaccine is recommended for children of both sexes aged 12–15 months but can also be given to non-immune adults. From October 1996 a second dose of MMR was recommended for children aged 4 years in order to prevent the re-accumulation of sufficient susceptible children to sustain future epidemics. Single antigen rubella vaccine continues to be given to girls aged 10–14 years, if they have not previously received MMR vaccine. In recent years several research papers have attributed an increase in autism to the introduction of the triple vaccine. This has led to a decreased public confidence in the vaccine. Detailed examination of the literature, and also the results of several clinical studies, have now indicated that there is no association between use of the triple vaccine and autism. This is backed up by over 20 years of successful deployment of the vaccine outside of the UK. Currently much effort is being made to restore confidence in the vaccine in order to avoid the lack of compliance leading to the occurrence of measles epidemics. 6.3 Tuberculosis Tuberculosis (TB) is a major cause of death and morbidity worldwide, particularly where poverty, malnutrition and poor housing prevail. Human infection is acquired by inhalation of Mycobacterium tuberculosis and M. bovis. Tuberculosis is primarily a disease of the lungs, causing chronic infection of the lower respiratory tract, but may spread to other sites or proceed to a generalized infection (miliary tuberculosis). Active disease can result either from a primary infection or from a subsequent reactivation of a quiescent infection. Following inhalation, the mycobacteria are taken up by alveolar macrophages where they survive and multiply. Circulating macrophages and lymphocytes, attracted to the site, carry the organism to local lymph nodes where a cell-mediated immune response is triggered. The host, unable to eliminate the pathogen, contains them within small granulomas or tubercles. If high numbers of mycobacteria are present then the cellular responses can result in tissue necrosis. The tubercles contain viable pathogens that may persist for the remaining life of the host. Reac147
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tivation of the healed primary lesions is thought to account for over two-thirds of all newly reported cases of the disease. The incidence of TB in the UK declined 10-fold between 1948 and 1987, since when just over 5000 new cases have been notified each year. Those most at risk include pubescent children, health service staff and individuals intending to stay for more than 1 month in countries where TB is endemic. A live vaccine is required to elicit protection against TB, as both antibody and cell-mediated immunity are required for protective immunity. Vaccination with BCG (bacille Calmette-Guérin), derived from an attenuated M. bovis strain, is commonly used in countries where TB is endemic. The vaccine was introduced in the UK in 1953 and was administered intradermally to children aged 13–14 years and to unprotected adults. Efficacy in the UK has been shown to be > 70% with protection lasting at least 15 years. In other countries, where the general state of health and well-being of the population is less than in the developed world, the efficacy of the vaccine has been shown to be significantly less than this. Because of the risks of adverse reaction to the vaccine by persons who have already been exposed to the disease a sensitivity test must be carried out before immunization with BCG. A Mantoux skin test assesses an individual’s sensitivity to a purified protein derivative (PPD) prepared from heat-treated antigens (tuberculin) extracted from M. tuberculosis. A positive test implies past infection or past, successful immunization. Those with strongly positive tests may have active disease and should be referred to a chest clinic. However, many people with active TB, especially disseminated TB, sero-convert from a positive to a negative skin test. Results of the skin test must therefore be interpreted with care. Much debate surrounds the use of BCG vaccine, a matter of some importance, considering that TB kills c. 3 million people annually and that drugresistant strains have emerged. While the vaccine has demonstrated some efficacy in preventing juvenile TB, it has little prophylactic effect against postprimary TB in those already infected. One solution is to bring forward the BCG immunization to include neonates. Immunization at 2–4 weeks of age will ensure that immunization precedes infection, 148
and also negates the requirement for a skin test. Passive acquired maternal antibody to TB is unlikely to interfere with the effectiveness of the immunization as immunity relates to a cell-mediated response. Alternative strategies involve improvement of the vaccine possibly through the introduction, into the BCG strain, of genes that encode protective antigens of M. tuberculosis. 6.4 Diphtheria, tetanus and pertussis (DTP) immunization Immunization against these unrelated diseases is considered together because the vaccines are nonliving and are often co-administered as a triple vaccine as part of the juvenile vaccination programme. 6.4.1 Diphtheria This is an acute, non-invasive infectious disease associated with the upper respiratory tract (Chapter 7). The incubation period is from 2 to 5 days although the disease remains communicable for up to 4 weeks. A low molecular weight toxin is produced which affects myocardium, nervous and adrenal tissues. Death results in 3–5% of infected children. Diphtheria immunization protects by stimulating the production of an antitoxin. This antitoxin will protect against the disease but not against infection of the respiratory tract. The immunogen is a toxoid, prepared by formaldehyde treatment of the purified toxin (Chapter 23) and administered while adsorbed to an adjuvant, usually aluminium phosphate or aluminium hydroxide. The primary course of diphtheria prophylaxis consists of three doses starting at 2 months of age and separated by an interval of at least 1 month. The immune status of adults may be determined by administration of Schick test toxin, which is essentially a diluted form of the vaccine. 6.4.2 Tetanus Tetanus is not an infectious disease but relates to the production of a toxin by germinating spores and vegetative cells of Clostridium tetani that might infect a deep puncture wound. The organism, which may be introduced into the wound from the soil,
Vaccination and immunization
grows anaerobically at such sites. The toxin is adsorbed into nerve cells and acts like strychnine on nerve synapses (Chapter 7). Tetanus immunization employs a toxoid and protects by stimulating the production of antitoxin. This antitoxin will neutralize the toxin as the organisms release it and before it can be adsorbed into nerves. As the toxin is produced only slowly after infection then the vaccine, which acts rapidly, may be used prophylactically in those non-immunized persons who have recently suffered a candidate injury. The toxoid, as with diphtheria toxoid, is formed by reaction with formaldehyde and is adsorbed onto an inorganic adjuvant. The primary course of tetanus prophylaxis consists of three doses starting at 2 months of age and is separated by an interval of at least 1 month. 6.4.3 Pertussis (whooping cough) Caused by the non-invasive respiratory pathogen Bordetella pertussis, whooping cough (Chapter 7) may be complicated by bronchopneumonia, repeated post-tussis vomiting leading to weight loss, and to cerebral hypoxia associated with a risk of brain damage. Until the mid-1970s the mortality from whooping cough was about one per 1000 notified cases with a higher rate for infants under 1 year of age. A full course of vaccine, which consists of a suspension of killed Bord. pertussis organisms (Chapter 23), gives complete protection in > 80% of recipients. The primary course of pertussis prophylaxis consists of three doses starting at 2 months of age and separated by an interval of at least 1 month. 6.4.4 DTP vaccine combinations and administration The primary course of DTP protection consists of three doses of a combined vaccine, each dose separated by at least 1 month and commencing not earlier than 2 months of age. In such combinations the pertussis component of the vaccine acts as an additional adjuvant for the toxoid elements. Monovalent pertussis and tetanus vaccines, and combined vaccines lacking the pertussis component (DT) are available. If pertussis vaccination is contraindi-
cated or refused then DT vaccine alone should be offered. The primary course of pertussis vaccination is considered sufficient to confer lifelong protection, especially as the mortality associated with disease declines markedly after infancy. The risks associated with tetanus and diphtheria infection persist throughout life. DT vaccination is therefore repeated before school entry, at 4–5 years of age, and once again at puberty. 6.5 Haemophilus influenzae type b (Hib) immunization Seven different capsular serotypes of Haemophilus influenzae type b (Hib) are associated with respiratory infection in young children. The most common presentation of these infections is as meningitis, frequently associated with bacteraemia. The sequelae, following Hib infection, include deafness, convulsions and intellectual impairment. The fatality rate is c. 4–5% with 8–11% of survivors having permanent neurological disorders. The disease, which is rare in children under 3 months, peaks both in its incidence and severity at 12 months of age. Infection is uncommon after 4 years of age. Before the introduction of Hib vaccination the incidence of the disease in the UK was estimated at 34 per 100 000. The vaccine utilizes purified preparations of the polysaccharide capsule of the major serotypes. Polysaccharides are poorly immunogenic and must be conjugated onto a protein carrier to enhance their efficacy. Hib vaccines are variously conjugated onto diphtheria and tetanus toxoids (above), group B meningococcal outer-membrane protein and a nontoxic derivative of diphtheria toxin (CRM197) and can now be mixed and co-administered with the DTP vaccine. Three doses of the vaccine are recommended, separated by 1 month. No reinforcement is recommended at 4 years of age as the risks from infection are negligible at this time. To avoid possible interactions, Hib vaccine was required, at its introduction, to be administered at a different body site to DTP. Sufficient evidence has now been gathered as to render this recommendation unnecessary. 6.6 Meningococcal immunization Meningococcal meningitis and septicaemia are sys149
Chapter 9
temic infections caused by strains of Neisseria meningitidis. There are at least 13 serotypes of which groups B and C are most common. In the UK type B accounts for approximately two-thirds of reported cases with type C accounting for the remaining third. Type C meningitis is not usually found in this country but is epidemic, particularly in the summer months, in other parts of the world. The most recent increases in meningococcal disease have been associated with group C infections in young adolescents. Group C meningitis is most common in the under 1-year-old group but its mortality is greatest in adolescents. Overall mortality from meningococcal disease is around 10%. The commonest manifestation is meningitis but in around 15–20% of cases septicaemia predominates. Mortality rates are much greater in septicaemic cases. At present there is no available vaccine for group B meningococcus but vaccines are available for groups A and C. As with the Hib vaccine the preparations are intended to invoke an immune responsiveness towards the polysaccharide component of the pathogens. Early vaccines were of a purified polysaccharide worked in adults and were of little efficacy in the most at-risk group of infants. The new MenC conjugate vaccine uses a similar technology to Hib and conjugates the polysaccharide components to a carrier protein (usually diphtheria toxin or tetanus toxoid). The resultant vaccine is effective in the very young and is suitable for infants. The vaccine is administered along with DTP and Hib at 2, 3 and 4 months. A single dose is sufficient to immunize individuals over 12 months of age and has been used to provide cover for teenagers, adolescents and young adults. Group C vaccine is available for those travelling to areas of the world where the infections are epidemic (e.g. Kenya).
7 Juvenile immunization schedule The timing of the various components of the juvenile vaccination programme is subject to continual review. In the 1960s, the primary course of DTP vaccination consisted of three doses given at 3, 6 and 12 months of age, together with OPV. This separation gave adequate time for the levels of induced 150
Table 9.1 Children’s immunization schedule for UK (1996) Vaccine
Age
Notes
BCG
Neonatal (1st month)
DTP, Hib, MenC Poliomyelitis MMR
lst dose 2 months 2nd dose 3 months 3rd dose 4 months 12–15 months
If not, at 13–14 years Primary course
Booster DT
3–5 years
Poliomyelitis MMR booster BCG Booster DT
3–5 years 3–5 years 10–14 years 13–18 years
Any time over 12 months 3 years after primary course
If not in infancy
antibody to decline between successive doses of the vaccines. Current recommendations (Table 9.1) accelerate the vaccination programme with no reductions in its efficacy. Thus, MMR vaccination has replaced separate measles and rubella prophylaxis and BCG vaccination may now be given at birth. DTP vaccination occurs at 2, 3 and 4 months to coincide with administration of Hib and MenC. It is imperative that as many individuals as possible benefit from the vaccination programme. Fewer visits to the doctor’s surgery translate into improved patient compliance and less likelihood of epidemic spread of the diseases in question. The current recommendations minimize the number of separate visits to the clinic while maximizing the protection generated.
8 Immunization of special risk groups While not recommended for routine administration, vaccines additional to those represented in the juvenile programme are available for individuals in special risk categories. These categories relate to occupational risks or risks associated with travel abroad. Such immunization protocols include those directed against cholera, typhoid, meningitis (type A), anthrax, hepatitis A and B, influenza, Japanese encephalitis, rabies, tick-borne encephalitis and yellow fever.
Vaccination and immunization
9 Further reading Mims, C. A., Nash, A. & Stephen, J. (2001) Mims’ Pathogenesis of Infectious Disease, 5th edn. Academic Press, London. Salisbury, D. M. & Begg, N. T. (1996) Immunisation Against
Infectious Disease. HMSO, London (updated every 4–5 years). Salyers, A. A. & Whitt, D. D. (1994) Bacterial Pathogenesis: A Molecular Approach. American Society for Microbiology Press, Washington.
151
Chapter 10
Types of antibiotics and synthetic antimicrobial agents A Denver Russell 1 Antibiotics 1.1 Definition 1.2 Sources 2 b-Lactam antibiotics 2.1 Penicillins and mecillinams 2.2 Cephalosporins 2.2.1 Structure-activity relationships 2.2.2 Pharmacokinetic properties 2.3 Clavams 2.4 1-Oxacephems 2.5 1-Carbapenems 2.5.1 Olivanic acids 2.5.2 Thienamycin and imipenem 2.6 1-Carbacephems 2.7 Nocardicins 2.8 Monobactams 2.9 Penicillanic acid derivatives 2.10 Hypersensitivity 3 Tetracycline group 3.1 Tetracyclines 3.2 Glycylcyclines 4 Rifamycins 5 Aminoglycoside-aminocyclitol antibiotics 6 Macrolides 6.1 Older members 6.2 Newer members 7 Lincosamides 8 Streptogramins 9 Polypeptide antibiotics 10 Glycopeptide antibiotics 10.1 Vancomycin 10.2 Teicoplanin
1 Antibiotics 1.1 Definition An antibiotic was originally defined as a substance, produced by one microorganism, which inhibited the growth of other microorganisms. The advent of 152
11 Miscellaneous antibacterial antibiotics 11.1 Chloramphenicol 11.2 Fusidic acid 11.3 Mupirocin (pseudomonic acid A) 12 Antifungal antibiotics 12.1 Griseofulvin 12.2 Polyenes 13 Synthetic antimicrobial agents 13.1 Sulphonamides 13.2 Diaminopyrimidine derivatives 13.3 Co-trimoxazole 13.4 Dapsone 13.5 Antitubercular drugs 13.6 Nitrofuran compounds 13.7 4-Quinolone antibacterials 13.8 Imidazole derivatives 13.9 Flucytosine 13.10 Synthetic allylamines 13.11 Synthetic thiocarbamates 13.12 Oxazolidinones 14 Antiviral drugs 14.1 Amantadines 14.2 Methisazone 14.3 Nucleoside analogues 14.4 Non-nucleoside compounds 14.4.1 Phosphonoacetic acid and sodium phosphonoformate 14.4.2 Protease inhibitors 14.5 Interferons 15 Drug combinations 16 Further reading
synthetic methods has, however, resulted in a modification of this definition and an antibiotic now refers to a substance produced by a microorganism, or to a similar substance (produced wholly or partly by chemical synthesis), which in low concentrations inhibits the growth of other microorganisms. Chloramphenicol was an early example.
Types of antibiotics and synthetic antimicrobial agents
Fig. 10.1 A, General structure of penicillins;
B, removal of side chain from benzylpenicillin; C, site of action of b-lactamases.
Antimicrobial agents such as sulphonamides (section 13.1) and the 4-quinolones (section 13.7), produced solely by synthetic means, are often referred to as antibiotics.
biotics that are valuable, or potentially important, antibacterial compounds. These will be considered briefly. 2.1 Penicillins and mecillinams
1.2 Sources There are three major sources from which antibiotics are obtained. 1 Microorganisms. For example, bacitracin and polymyxin are obtained from some Bacillus species; streptomycin, tetracyclines, etc. from Streptomyces species; gentamicin from Micromonospora purpurea; griseofulvin and some penicillins and cephalosporins from certain genera (Penicillium, Acremonium) of the family Aspergillaceae; and monobactams from Pseudomonas acidophila and Gluconobacter species. Most antibiotics in current use have been produced from Streptomyces spp. 2 Synthesis. Chloramphenicol is now usually produced by a synthetic process. 3 Semisynthesis. This means that part of the molecule is produced by a fermentation process using the appropriate microorganism and the product is then further modified by a chemical process. Many penicillins and cephalosporins (section 2) are produced in this way. In addition, it has been suggested that (a) some bacteriophages (Chapter 5) might have an important role to play in the chemotherapy of bacterial infections, and (b) plant products might prove to be a potentially fruitful source of new antimicrobial agents.
2 b-Lactam antibiotics There are several different types of b-lactam anti-
The penicillins (general structure, Fig. 10.1A) may be considered as being of the following types. 1 Naturally occurring. For example, those produced by fermentation of moulds such as Penicillium notatum and P. chrysogenum. The most important examples are benzylpenicillin (penicillin G) and phenoxymethylpenicillin (penicillin V). 2 Semisynthetic. In 1959, scientists at Beecham Research Laboratories succeeded in isolating the penicillin ‘nucleus’, 6-aminopenicillanic acid (6-APA; Fig. 10.1A: R represents H). During the commercial production of benzylpenicillin, phenylacetic (phenylethanoic) acid (C6H5.CH2.COOH) is added to the medium in which the Penicillium mould is growing (see Chapter 22). This substance is a precursor of the side-chain (R; see Fig. 10.2) in benzylpenicillin. Growth of the organism in the absence of phenylacetic acid led to the isolation of 6-APA; this has a different RF value from benzylpenicillin, which allowed it to be detected chromatographically. A second method of producing 6-APA came with the discovery that certain microorganisms produce enzymes, penicillin amidases (acylases), which catalyse the removal of the side-chain from benzylpenicillin (Fig. 10.1B). Acylation of 6-APA with appropriate substances results in new penicillins being produced that differ only in the nature of the side-chain (Table 10.1; Fig. 10.2). Some of these penicillins have considerable activity against Gram-negative as well as Grampositive bacteria, and are thus broad-spectrum 153
Types of antibiotics and synthetic antimicrobial agents
antibiotics. Pharmacokinetic properties may also be altered. The sodium and potassium salts are very soluble in water but they are hydrolysed in solution, at a
temperature-dependent rate, to the corresponding penicilloic acid (Fig. 10.3A), which is not antibacterial. Penicilloic acid is produced at alkaline pH or (via penicillenic acid; Fig. 10.3B) at neutral pH, but
Table 10.1 The penicillins and mecillinams Stability to b-lactamases from
Activity versus
Penicillin
Orally effective
Staph. aureus
Gram -ve
Gram -ve*
Ps. aeruginosa
Ester
Hydrolysed after absorption
1 2 3 4 5 6 7 8 9 10 11 12 13
+ + + + + + + + +
+ + + + + -
+ + + + + + + + +
+ + + + + + +
+ + + + +
+ +
+ +
+ + + +
NR NR
V V
+ + + + + + + +
+ + + -
+ + + +
+ + + +
14 15 16 17 18 19 20 21
Benzylpenicillin Phenoxymethylpenicillin Methicillin Oxacillin Cloxacillin Flucloxacillin Ampicillin Amoxycillin Carbenicillin Ticarcillin Temocillin Carfecillin Carbenicillin Indanyl carbenicillin esters (carindacillin) Pivampicillin Ampicillin Talampicillin esters Bacampicillin Piperacillin Substituted Azlocillin ampicillins Mezlocillin 6-b-amidinoMecillinam Pivmecillinam penicillins
}
}
}
}
* Except Ps. aeruginosa. All penicillins show some degree of activity against Gram-negative cocci. +, applicable. -, inapplicable. NR, not relevant: mecillinam and pivmecillinam have no effect on Gram-positive bacteria: V, variable. Note: 1 Esters give high urinary levels. 2 Hydrolysis of these esters by enzyme action after absorption from the gut mucosa gives rapid and high blood levels. 3 For additional information on resistance to b-lactamase inactivation, see Chapter 13. 4 In general, all penicillins are active against Gram-positive bacteria, although this may depend on the resistance of the drug to b-lactamase (see column 3): thus, benzylpenicillin is highly active against strains of Staphylococcus aureus which do not produce b-lactamase, but is destroyed by b-lactamase-producing strains. 5 Temocillin number (11) is less active against Gram-positive bacteria than ampicillin or the ureidopenicillins (substituted ampicillins). 6 Not all of the antibiotics listed above are currently available, but are included to illustrate the stages in development of the penicillins.
䉳
Fig. 10.2 Examples of the side-chain R in various penicillins and mecillinams (the numbers 1–21 correspond to those in Table
10.1). Numbers 20 (mecillinam) and 21 (pivmecillinam) are 6-b-amidinopenicillanic acids (mecillinams). Number 11 (temocillin) has a methoxy (—OCH3) group at position 6a; this confers high b-lactamase stability on the molecule.
155
Chapter 10
Fig. 10.3 Degradation products of benzylpenicillin in solution: A, penicilloic acid; B, penicillenic acid; C, penillic acid.
at acid pH a molecular re-arrangement occurs, giving penillic acid (Fig. 10.3C). Instability in acid medium logically precludes oral administration, as the antibiotic may be destroyed in the stomach; for example at pH 1.3 and 35°C methicillin has a halflife of only 2–3 minutes and is therefore not administered orally, whereas ampicillin, with a half-life of 600 minutes, is obviously suitable for oral use. Benzylpenicillin is rapidly absorbed and rapidly excreted. However, certain sparingly soluble salts of benzylpenicillin (benzathine, benethamine and procaine) slowly release penicillin into the circulation over a period of time, thus giving a continuous high concentration in the blood. Pro-drugs (e.g. carbenicillin esters, ampicillin esters; Fig. 10.2, Table 10.1) are hydrolysed by enzyme action after absorption from the gut mucosa to produce high blood levels of the active antibiotic, carbenicillin and ampicillin, respectively. Several bacteria produce an enzyme, b-lactamase (penicillinase; see Chapter 13), which may inactivate a penicillin by opening the b-lactam ring, as in Fig. 10.1C. However, some penicillins (Table 10. l) are considerably more resistant to this enzyme than others, and consequently may be extremely valuable in the treatment of infections caused by b-lactamase-producing bacteria. In general, the penicillins are active against Gram-positive bacteria; some members (e.g. amoxycillin) are also effective against Gram-negative bacteria, although not Pseudomonas aeruginosa, whereas others (e.g. ticarcillin) are also active against this organism. In 156
particular, substituted ampicillins (piperacillin and the ureidopenicillins, azlocillin and mezlocillin) appear to combine the properties of ampicillin and carbenicillin. Temocillin is the first penicillin to be completely stable to hydrolysis by b-lactamases produced by Gram-negative bacteria. The 6-b-amidinopenicillanic acids, mecillinam and its ester pivmecillinam, have unusual antibacterial properties, as they are active against Gramnegative but not Gram-positive organisms. 2.2 Cephalosporins In the 1950s, a species of Cephalosporium (now known as Acremonium: see Chapter 22) isolated near a sewage outfall off the Sardinian coast was studied at Oxford and found to produce the following antibiotics. 1 An acidic antibiotic, cephalosporin P (subsequently found to have a steroid-like structure). 2 Another acidic antibiotic, cephalosporin N (later shown to be a penicillin, as its structure was based on 6-APA). 3 Cephalosporin C, obtained during the purification of cephalosporin N; this is a true cephalosporin, and from it has been obtained 7aminocephalosporanic acid (7-ACA; Fig. 10.4), the starting point for new cephalosporins. Cephalosporins consist of a six-membered dihydrothiazine ring fused to a b-lactam ring. Thus, the cephalosporins (D3-cephalosporins) are structurally related to the penicillins (section 2.1). The
Types of antibiotics and synthetic antimicrobial agents R3 1
R
NH
7 8
O
6 N5
S 1 4
2 3
CH2
R2
–
COO 1
Cephalosporin
R
H
7-ACA Cephacetrile
2
R
N
O.CO.CH3
C.CH2.CO
Cephaloridine CH2CO
S
CH.CO
Cephalexin
O.CO.CH3 + N
H
NH2 Cefuroxime
C.CO O
O.CO.NH2
N.O.CH3 Cefoxitin S Cefaclor
CH2CO
O.CO.NH2
CH.CO
Cl (attached directly to ring at 3)
NH2
Cephalothin
CH2CO
S
Cephapirin
N
OCOCH3
OCOCH3
S.CH2CO
O
Cefsulodin N+
CH.CO–
C NH2
SO3Na N Cefazolin
N N
CH2CO
N
Cephradine
N
N S
CH3
S
CH.CO
H
NH2 Cefamandole
CH.CO OH
N
N
S N
N
CH3
Fig. 10.4 (above and overleaf ) General structure of cephalosporins and examples of side-chains R and R2. (R3 is —OCH3 in
cefoxitin and cefotetan and — H in other members.) Cephalosporins containing an ester group at position 3 are liable to attack by esterases in vivo.
position of the double bond in D3-cephalosporins is important, as D2-cephalosporins (double bond between 2 and 3) are not antibacterial irrespective of the composition of the side-chains.
2.2.1 Structure-activity relationships The activity of cephalosporins (and other blactams) against Gram-positive bacteria depends on antibiotic affinity for penicillin-sensitive 157
Chapter 10 R1
Cephalosporin
R2
HOOC O.CO.CH3
CH.(CH2)3.CO
Cephalosporin C H2N
N
S
H2N Ceftizoxime
O.CO.CH3
C.CO
N
Cefotaxime
OCH3 H (instead of 2 CH2 R)
C.CO
N
N
S
H 2N
OCH3 CH3 C.CO
N
Ceftriaxone
N
S
H2N
C.CO
N
Ceftazidine
S
OCH3
ONa
N
O
+ N
N
S
H2N
N N
O CH3
C
CH3
COONa
Cefotetan
N
S
H2NOC C
CH
C
N
S
CO
N N
S
HOOC
CH3 Cefoperazone
CH
HO
CO N
NH
O
O
S
N
N N
O
C C2H5
N
N
CH3
C.CO
N
CH
CH2
O
CH3 (At 4: COOCH(CH3)OCO2 CH(CH3)2)
Cefixime S
H 2N
N O CH2COOH C.CO
N Cefpodoxime S
H 2N
N.OCH3 H (no –CH2)
C.CO
N Ceftibuten S
H2N
CH CH2 COOH
C.CO
N
Cefpirome
S
H2N
N+
NH2
N OCH3 C.CO
N
Cefepime H2N
S
+
N
N OCH3
Fig. 10.4 Continued
H3C
Types of antibiotics and synthetic antimicrobial agents
enzymes (PSEs) detected in practice as penicillinbinding proteins (PBPs). Resistance results from altered PBPs or, more commonly, from b-lactamases. Activity against Gram-negative bacteria depends upon penetration of b-lactams through the outer membrane, resistance to b-lactamases found in the periplasmic space and binding to PBPs. (For further information on mechanisms of action and bacterial resistance, see Chapters 12 and 13). Modifications of the cephem nucleus (Fig. 10.4) at 7a, i.e. R3, by addition of methoxy groups increase b-lactamase stability but decrease activity against Grampositive bacteria because of reduced affinity for PBPs. Side-chains containing a 2-aminothiazolyl group at R1, e.g. cefotaxime, ceftizoxime, ceftriaxone and ceftazidime, yield cephalosporins with enhanced affinity for PBPs of Gram-negative bacteria and streptococci. An iminomethoxy group (—C=N.OCH3) in, for example, cefuroxime provides b-lactamase stability against common plasmid-mediated b-lactamases. A propylcarboxy group ((CH3)2—C—COOH) in, for example, ceftazidime increases b-lactamase resistance and also provides activity against Ps. aeruginosa, whilst at the same time reducing b-lactamase induction capabilities. Further examples of the interplay of factors in antibacterial activity are demonstrated by the following findings. 1 7a-methoxy substitution of cefuroxime, cefamandole and cephapirin produces reduced activity against E. coli because of a lower affinity for PBPs; 2 similar substitution of cefoxitin produces enhanced activity against E. coli because of greater penetration through the outer membrane of the organism. In cephalosporins susceptible to b-lactamases, opening of the b-lactam ring occurs with concomitant loss of the substituent at R2 (except in cephalexin, where R2 represents H; see Fig. 10.4). This is followed by fragmentation of the molecule. Provided that they are not inactivated by blactamases, the cephalosporins generally have a broad spectrum of activity, although there may be a wide variation. Haemophilus influenzae, for example, is particularly susceptible to cefuroxime; see also Table 10.2.
2.2.2 Pharmacokinetic properties Pharmacokinetic properties of the cephalosporins depend to a considerable extent on their chemical nature, e.g. the substituent R2. The 3acetoxymethyl compounds such as cephalothin, cephapirin and cephacetrile are converted in vivo by esterases to the antibacterially less active 3hydroxymethyl derivatives and are excreted partly as such. The rapid excretion means that such cephalosporins have a short half-life in the body. Replacement of the 3-acetoxymethyl group by a variety of groups has rendered other cephalosporins much less prone to esterase attack. For example, cephaloridine has an internally compensated betaine group at position 3 (R2) and is metabolically stable. Cephalosporins such as the 3-acetoxymethyl derivatives described above, cephaloridine and cefazolin are inactive when given orally. For good oral absorption, the 7-acyl group (R1) must be based on phenylglycine and the amino group must remain unsubstituted. The R2 substituent must be small, non-polar and stable; a methyl group is considered desirable but might decrease antibacterial activity. Earlier examples of oral cephalosporins are provided by cephalexin, cefaclor and cephradine (Table 10.2). Newer oral cephalosporins such as cefixime, cefpodoxime and ceftibuten show increased stability to b-lactamases produced by Gram-negative bacteria. Like cefuroxime axetil (also given orally), cefpodoxime is an absorbable ester. During absorption, esterases remove the ester side-chain, liberating the active substance into the blood. Cefixime and ceftibuten are non-ester drugs characterized by activity against Gram-positive and Gram-negative bacteria, although Ps. aeruginosa is resistant. Parenterally administered cephalosporins that are metabolically stable and that are resistant to many types of b-lactamases include cefuroxime, cefamandole, cefotaxime and cefoxitin, which has a 7b-methoxy group at R2. Injectable cephalosporins with anti-pseudomonal activity include ceftazidime, cefsulodin and cefoperazone. Side-chains of the various cephalosporins, including those most recently developed, are presented in Fig. 10.4 and a summary of the properties of these antibiotics in Table 10.2. 159
Chapter 10 Table 10.2 The cephalosporins*
Staphylococci†
Staphylococcal b-lactamase Streptococci‡
Enterobacteria
Enterobacterial b-lactamases
Neisseria
Haemophilus
Ps. aeruginosa
Properties
++
++
+
V
V
+
(+)
R
+
++
++
V
V
++
++
R
++ ++ ++
++ ++ +
++ ++ +
V V V
V V V
++ ++ +
++ ++ (+)
R R R
++
++
++
++
++
++
++
R
Cefotaxime, ceftazidime, ceftizoxime, ceftriaxone (also the oxacephem, latamoxef, section 2.4) Cefoperazone Cefsulodin
++
++
+++
+++
+++
+++
+++
R (ceftazidime +++)
++ (+)
++ ++
+ (+)
V
V
++
++ R
++ +++
Cefotetan
(+)
+++
+++
Group
Examples
Oral cephalosporins
Cephalexin, cephradine, cefaclor, cefadroxil Cefixime, ceftibuten Cefuroxime axetil Cefpodoxime axetil Cephaloridine, cephalothin, cephacetrile, cefazolin Cefuroxime, cefoxitin, cefamandole
Injectable cephalosporins (b-lactamase susceptible) Injectable cephalosporins (improved b-lactamase stability) Injectable cephalosporins (still higher b-lactamase stability)
Injectable cephalosporins (anti-pseudomonal activity) Injectable cephalosporins (other)
R
Comment
Newer oral cephalosporins Absorbable ester Absorbable ester
Cefoxitin shows activity against Bacteroides fragilis Latamoxef has high activity against B. fragilis
Inhibits B. fragilis
* Early cephalosporins were spelt with ‘ph’, more recently with ‘f’. † Methicillin-resistant Staph. aureus (MRSA) strains are resistant to cephalosporins. ‡ Enterococci are resistant to cephalosporins. +++, excellent; ++, good; +, fair; (+), poor; R, resistant; V, variable.
2.3 Clavams The clavams differ from penicillins (based on the penam structure) in two respects, namely the replacement of sulphur in the penicillin thiazolidine 160
ring (Fig. 10.1) with oxygen in the clavam oxazolidine ring (Fig. 10.5A) and the absence of the sidechain at position 6. Clavulanic acid, a naturally occurring clavam isolated from Streptomyces clavuligerus, has poor antibacterial activity but is a
Types of antibiotics and synthetic antimicrobial agents A CH2OH
B OCH3
O CH
HO
CO
N H
N
N
COOH
O
O
COONa O
CH2
5 4 N
6
1
2 3
H
O
S
N N
COONa
D
C
CH3 H
N
N
CH3 H
R
NaO3SO N O
CO3Na
E CH3 F CH3
S
HO
NH2 (–N=CHNH2 in imipenem)
N
OH H
H
CH3
CH3
H
H
H3C
O
COO–
COOH Na+
N O COO–
R¢
NH
N
CH3 S
H
CH3
S
G OH
CON·
COOH
O
NH N+ H2
O
I
R3 NH
7 8
CH
6 1 2 5 3 N 4
CH O
CH2
O
CH
R2
7
NH2
3 N Cl COOH
COO
Fig. 10.5 A, clavulanic acid; B, latamoxef; C, 1-carbapenems; D, olivanic acid (general structure); E, thienamycin;
F, meropenem; G, ertapenem; H, 1-carbacephems; I, loracarbef.
potent inhibitor of staphylococcal b-lactamase and of most types of b-lactamases produced by Gramnegative bacteria, especially those with a ‘penicillinase’ rather than a ‘cephalosporinase’ type of enzyme action. A significant development in chemotherapy has been the introduction into clinical practice of a combination of clavulanic acid with a broadspectrum, but b-lactamase-susceptible, penicillin,
amoxycillin. The spectrum of activity has been extended to include Ps. aeruginosa by combining clavulanic acid with the b-lactamase-susceptible penicillin, ticarcillin. 2.4 1-Oxacephems In the 1-oxacephems, for example latamoxef (moxalactam, Fig. 10.5B), the sulphur atom in the 161
Chapter 10
dihydrothiazine cephalosporin ring system is replaced by oxygen. This would tend to make the molecule chemically less stable and more susceptible to inactivation by b-lactamases. The introduction of the 7-a-methoxy group (as in cefoxitin, Fig. 10.4), however, stabilizes the molecule. Latamoxef is a broad-spectrum antibiotic with a high degree of stability to most types of b-lactamases, and is highly active against the anaerobe B. fragilis.
2.6 1-Carbacephems In the 1-carbacephems (Fig. 10.5H), the sulphur in the six-membered dihydrothiazine ring of the cephalosporins (based on the cephem structure, see Fig. 10.4) is replaced by carbon. Loracarbef (Fig. 10.5I) is a newer oral carbacephem which is highly active against Gram-positive bacteria, including staphylococci.
2.5 1-Carbapenems
2.7 Nocardicins
The 1-carbapenems (Fig. 10.5C) comprise a family of fused b-lactam antibiotics. They are analogues of penicillins or clavams, the sulphur (penicillins) or oxygen (clavams) atom being replaced by carbon. Examples are the olivanic acids (section 2.5.1) and thienamycin and imipenem (section 2.5.2).
The nocardicins (A to G) have been isolated from a strain of Nocardia and comprise a novel group of blactam antibiotics (Fig. 10.6A). Nocardicin A is the most active member, and possesses significant activity against Gram-negative but not Gram-positive bacteria.
2.5.1 Olivanic acids
2.8 Monobactams
The olivanic acids (general structure, Fig. 10.5D) are naturally occurring b-lactam antibiotics which have, with some difficulty, been isolated from culture fluids of Strep. olivaceus. They are broadspectrum antibiotics and are potent inhibitors of various types of b-lactamases.
The monobactams are monocyclic b-lactam antibiotics produced by various strains of bacteria. A novel nucleus, 3-aminomonobactamic acid (3AMA, Fig. 10.6B), has been produced from naturally occurring monobactams and from 6-APA. Several monobactams have been tested and one (aztreonam, Fig. 10.6C) has been shown to be highly active against most Gram-negative bacteria and to be stable to most types of b-lactamases. It is not destroyed by staphylococcal b-lactamases but is inactive against all strains of Staph. aureus tested. Bacteroides fragilis, a Gram-negative anaerobe, is resistant to aztreonam, probably by virtue of the b-lactamase it produces, and this conclusion is supported by the finding that a combination of the monobactam with clavulanic acid (section 2.3) is ineffective against this organism.
2.5.2 Thienamycin and imipenem Thienamycin (Fig. 10.5E) is a broad-spectrum blactam antibiotic with high b-lactamase resistance. Unfortunately, it is chemically unstable, although the N-formimidoyl derivative, imipenem, overcomes this defect. Imipenem (Fig. 10.5E) is stable to most b-lactamases but is readily hydrolysed by kidney dehydropeptidase and is administered with a dehydropeptidase inhibitor, cilastatin. Meropenem, marketed more recently, is more stable than imipenem to this enzyme and may thus be administered without cilastatin. Its chemical structure is depicted in Fig. 10.5F. Ertapenem (Fig. 10.5G) has properties similar to those of meropenem but affords the additional advantage of once-daily dosing.
162
2.9 Penicillanic acid derivatives Penicillanic acid derivatives are synthetically produced b-lactamase inhibitors. Penicillanic acid sulphone (Fig. 10.6D) protects ampicillin from hydrolysis by staphylococcal b-lactamase and some, but not all, of the b-lactamases produced by Gram-negative bacteria, but is less potent than
Types of antibiotics and synthetic antimicrobial agents
Fig. 10.6 A, Nocardicin A; B, 3-aminomonobactamic acid (3-AMA); C, aztreonam; D, penicillanic acid sulphone (sodium salt);
E, b-bromopenicillanic acid (sodium salt); F, tazobactam; G, sulbactam.
clavulanic acid. b-Bromopenicillanic acid (Fig. 10.6E) inhibits some types of b-lactamases. Tazobactam (Fig. 10.6F) is a penicillanic acid sulphone derivative marketed as a combination with piperacillin. Alone it has poor intrinsic antibacterial activity but is comparable to clavulanic acid in inhibiting b-lactamase activity. Sulbactam (Fig. 10.6G) is a semisynthetic 6desaminopenicillin sulphone structurally related to tazobactam. Not only is it an effective inhibitor of many b-lactamases but it is also active alone against certain Gram-negative bacteria. It is used clinically in combination with ampicillin. 2.10 Hypersensitivity Some types of allergic reaction, for example immediate or delayed-type skin allergies, serum sicknesslike reactions and anaphylactic reactions, may occur in a proportion of patients given penicillin
treatment. There is some, but not complete, crossallergy with cephalosporins. Contaminants of high molecular weight (considered to have arisen from mycelial residues from the fermentation process) may be responsible for the induction of allergy to penicillins; their removal leads to a marked reduction in the antigenicity of the penicillin. It has also been found, however, that varying amounts of a non-protein polymer (of unknown source) may also be present in penicillin and that this also may be antigenic. The interaction of a non-enzymatic degradation product, d-benzylpenicillenic acid (formed by cleavage of the thiazolidine ring of benzylpenicillin in solution; see Fig. 10.3B), with sulphydryl or amino groups in tissue proteins, to form haptenprotein conjugates, is also of importance. In particular, the reaction between d-benzylpenicillenic acid and the e-amino group of lysine (a,e-diamino-ncaproic acid, NH2(CH2)4.CH(NH2).COOH) residues 163
Chapter 10
Fig. 10.7 Tetracycline antibiotics: 1, oxytetracycline; 2, chlortetracycline; 3, tetracycline; 4, demethylchlortetracycline; 5, doxycycline; 6, methacycline; 7, clomocycline; 8, minocycline; 9, thiacycline (a thiatetracycline with a sulphur atom at 6).
is to be noted, because these d-benzylpenicilloyl derivatives of tissue proteins function as complete penicillin antigens.
3 Tetracycline group 3.1 Tetracyclines There are several clinically important tetracyclines, characterized by four cyclic rings (Fig. 10.7). They consist of a group of antibiotics obtained as byproducts from the metabolism of various species of Streptomyces, although some members may now be thought of as being semisynthetic. Thus, tetracycline (by catalytic hydrogenation) and clomocycline are obtained from chlortetracycline, which is itself produced from Strep. aureofaciens. Methacycline is obtained from oxytetracycline (produced from Strep. rimosus) and hydrogenation of methacycline gives doxycycline. Demethylchlortetracycline is produced by a mutant strain of Strep. 164
aureofaciens. Minocycline is a derivative of tetracycline. The tetracyclines are broad-spectrum antibiotics, i.e. they have a wide range of activity against Gram-positive and Gram-negative bacteria. Ps. aeruginosa is less sensitive, but is generally susceptible to tetracycline concentrations obtainable in the bladder. Resistance to the tetracyclines (see also Chapter 13) develops relatively slowly, but there is cross-resistance, i.e. an organism resistant to one member is usually resistant to all other members of this group. However, tetracycline-resistant Staph. aureus strains may still be sensitive to minocycline. Suprainfection (‘overgrowth’) with naturally tetracycline-resistant organisms, for example Candida albicans and other yeasts, and filamentous fungi, affecting the mouth, upper respiratory tract or gastrointestinal tract, may occur as a result of the suppression of tetracycline-susceptible microorganisms. Thiatetracyclines contain a sulphur atom at position 6 in the molecule. One derivative, thiacycline,
Types of antibiotics and synthetic antimicrobial agents A
N(CH3)2 OH O 9
(CH3)2N
CONH2
N H
OH OH
OH
O N(CH3)2
(CH3)2N
B
OH
Fig. 10.8 Structures of two tetracycline
analogues, which are members of the new glycylcycline group of antibiotics: A, N,N-dimethylglycylamido-6-demethyl-6deoxytetracycline; B, N,Ndimethylglycylamidominocycline.
O
O
7
(CH3)2N
is more active than minocycline against tetracycline-resistant bacteria. Despite toxicity problems affecting its possible clinical use, thiacycline could be the starting point in the development of a new range of important tetracycline-type antibiotics. The tetracyclines are no longer used clinically to the same extent as they were in the past because of the increase in bacterial resistance. 3.2 Glycylcyclines The glycylcyclines (Fig. 10.8) represent a new group of tetracycline analogues. They are novel tetracyclines substituted at the C-9 position with a dimethylglycylamido side-chain. They possess activity against bacteria that express resistance to the older tetracyclines by an efflux mechanism (Chapter 13).
4 Rifamycins Rifamycins A to E have been described. From rifamycin B are produced rifamide (rifamycin B diethylamide) and rifamycin SV, which is one of the most useful and least toxic of the rifamycins. Rifampicin (Fig. 10.9), a bactericidal antibiotic, is active against Gram-positive bacteria (including Mycobacterium tuberculosis) and some Gram-
9 CONH2
N H
OH OH
O
OH
O
negative bacteria (but not Enterobacteriaceae or pseudomonads). It has been found to have a greater bactericidal effect against M. tuberculosis than other antitubercular drugs, is active orally, penetrates well into cerebrospinal fluid and is thus of use in the treatment of tuberculous meningitis (see also section 13.5). Rifampicin possesses significant bactericidal activity at very low concentrations against staphylococci. Unfortunately, resistant mutants may arise very rapidly, both in vitro and in vivo. It has thus been recommended that rifampicin should be combined with another antibiotic, e.g. vancomycin, in the treatment of staphylococcal infections. Rifabutin may be used in the prophylaxis of M. avium complex infections in immunocompromised patients and in the treatment, with other drugs, of pulmonary tuberculosis and non-tuberculous mycobacterial infections.
5 Aminoglycoside-aminocyclitol antibiotics Aminoglycoside antibiotics contain amino sugars in their structure. Deoxystreptamine-containing members are neomycin, framycetin, gentamicin, kanamycin, tobramycin, amikacin, netilmicin and sisomicin. Both streptomycin and dihydrostreptomycin contain streptidine, whereas the aminocycli165
Chapter 10
Fig. 10.9 Rifampicin.
tol spectinomycin has no amino sugar. Examples of chemical structures are provided in Fig. 10.10. Streptomycin (Fig. 10.10A) was isolated by Waksman in 1944, and its activity against M. tuberculosis ensured its use as a primary drug in the treatment of tuberculosis. Unfortunately, its ototoxicity and the rapid development of resistance have tended to modify its usefulness, and although it still remains a useful drug against tuberculosis it is usually used in combination with other antibiotics. Streptomycin also shows activity against other types of bacteria, for example against various Gram-negative bacteria and some strains of staphylococci. Dihydrostreptomycin has a similar antibacterial action but is more toxic. Kanamycin (a complex of three antibiotics, A, B and C; Fig. 10.10B) is active in low concentrations against various Gram-positive (including penicillin-resistant staphylococci) and Gram-negative bacteria. It is a recognized second-line drug in the treatment of tuberculosis. Gentamicin (a mixture of three components, C1, C1a and C2; Fig. 10.10C) is active against many strains of Gram-positive and Gram-negative bacteria, including some strains of Ps. aeruginosa. Its activity is greatly increased at pH values of about 8. It is often administered in conjunction with a blactam to delay the development of resistance. Gentamicin is the most important aminoglycoside antibiotic, is the aminoglycoside of choice in the UK and is widely used for treating serious infections. As with other members of this group, side-effects 166
are dose-related, dosage must be given with care, plasma levels should be monitored and treatment should not normally exceed 7 days. Paromomycin finds special use in the treatment of intestinal amoebiasis (it is amoebicidal against Entamoeba histolytica) and of acute bacillary dysentery. Neomycin is poorly absorbed from the alimentary tract when given orally, and is usually used in the form of lotions and ointments for topical application against skin and eye infections. Framycetin consists of neomycin B with a small amount of neomycin C, and is usually employed locally. A desirable property of newer aminoglycoside antibiotics is increased antibacterial activity against resistant strains, especially improved stability to aminoglycoside-modifying enzymes (Chapter 13). Alteration in the 3¢-position of kanamycin B (Fig. 10.10B) to give 3¢-deoxykanamycin B (tobramycin) changes the activity spectrum. Amikacin (Fig. 10.10D) has a substituted aminobutyryl in the amino group at position 1 in the 2deoxystreptamine ring and this enhances its resistance to some, but not all, types of aminoglycosidemodifying enzymes, as it has fewer sites of modification. Netilmicin (N-ethylsisomicin) is a semisynthetic derivative of sisomicin but is less susceptible than sisomicin to some types of bacterial enzymes. The most important of these antibiotics are amikacin, tobramycin, netilmicin and especially gentamicin.
6 Macrolides 6.1 Older members The macrolide antibiotics are characterized by possessing molecular structures that contain large (12–16-membered) lactone rings linked through glycosidic bonds with amino sugars. The most important members of this group are erythromycin (Fig. 10.11), oleandomycin, triacetyloleandomycin and spiramycin. Erythromycin is active against most Gram-positive bacteria, Neisseria, H. influenzae and Legionella pneumophila, but not against the Enterobacteriaceae; its activity is
Types of antibiotics and synthetic antimicrobial agents
Fig. 10.10 Some aminoglycoside antibiotics: A, streptomycin; B, kanamycins; C, gentamicins; D, amikacin.
pH-dependent, increasing with pH up to about 8.5. Erythromycin estolate is more stable than the free base to the acid of gastric juice and is thus employed for oral use. The estolate produces higher and more prolonged blood levels and distributes into some
tissues more efficiently than other dosage forms. In vivo, it hydrolyses to give the free base. Staphylococcus aureus is less sensitive to erythromycin than are pneumococci or haemolytic streptococci, and there may be a rapid development 167
Chapter 10
Fig. 10.11 Erythromycins:
erythromycin is a mixture of macrolide antibiotics consisting largely of erythromycin A.
of resistance, especially of staphylococci, in vitro. However, in vivo with successful short courses of treatment, resistance is not usually a serious clinical problem. On the other hand, resistance is likely to develop when the antibiotic is used for long periods. Oleandomycin, its ester (triacetyloleandomycin) and spiramycin have a similar range of activity to erythromycin but are less potent. Resistance develops only slowly in clinical practice. However, crossresistance may occur between all four members of this group. 6.2 Newer members The new macrolides are semisynthetic molecules that differ from the original compounds in the substitution pattern of the lactone ring system (Table 10.3, Figs 10.12 and 10.13). Roxithromycin has similar in vitro activity to erythromycin but enters leucocytes and macrophages more rapidly with higher concentrations in the lysosomal component of the phagocytic cells. It is likely to become an important drug against Legionella pneumophila. Clarithromycin and azithromycin are also of value.
7 Lincosamides Lincomycin and clindamycin (Fig. 10.14A, B) are 168
Table 10.3 Newer macrolide derivatives of erythromycin Lactone ring structure 14-membered
15-membered
Example Erythromycin Roxithromycin Clarithromycin Dirithromycin Azithromycin
Derivative of erythromycin — Methoxy-ethoxymethyloxine Methyl Oxazine Deoxo-aza-methyl-homo
active against Gram-positive cocci, except Enterococcus faecalis. Gram-negative cocci tend to be less sensitive and enterobacteria are resistant. Although not related structurally to the macrolides (section 6), the lincosamides have a similar mechanism of action. Cross-resistance may occur between lincosamides, streptogramins and macrolides, but some erythromycin-resistant organisms may be sensitive to lincosamides.
8 Streptogramins Pristinamycin and virginiamycin are two streptogramins that have been long been known. Pristinamycin is a mixture of synergistic components, pristinamycins I and II, the former being a macrolide and the latter a depsipeptide.
Types of antibiotics and synthetic antimicrobial agents CH3
A CH3OCH2CH2OCH2O CH3
N
CH3
N CH3
CH3
OH
OH
CH3 OH
HO
CH3 O
HO
CH3
CH3
OH CH3CH2
CH3
OH
CH3
O
O
O
CH3CH2
N(CH3)2 CH3
O
O
N(CH3)2 O
CH3
O OCH3
O
O
CH3
OCH3 CH3
OH
Fig. 10.13 Structure of azithromycin (15-membered
OH
CH3 B
CH3
CH3
O
CH3
O
macrolide).
O CH3
CH3
OH
OCH3
CH3
CH3
OH
HO
CH3 O
CH3CH2
O
O
N(CH3)2 O
CH3
O OCH3 CH3
CH3
O CH3
OH
The MLS (macrolides, lincosamides, streptogramins) group of antibiotics all inhibit protein synthesis by binding to the 50S ribosomal subunit. Resistance mechanisms specific to individual members occur but resistance to all may be conferred by a single mechanism that involves 23S rRNA. However, it is claimed that the quinupristin-dalfopristin combination does not demonstrate cross-resistance to other antibiotics within the MLS group or to other antibiotics.
Fig. 10.12 Examples of newer 14-membered macrolides:
A, roxithromycin; B, clarithromycin.
Two important new streptogramins are quinupristin (Fig. 10.15A) and dalfopristin (Fig. 10.15B), which are derivatives of pristinamycin I and IIA, respectively. Individually, the two components are bacteristatic but in combination they act synergistically to produce a bactericidal effect by inhibiting early (dalfopristin) and late (quinupristin) phases of bacterial protein synthesis. These two antibiotics are used intravenously in combination (ratio 30 : 70) for the treatment of serious or lifethreatening infections associated with vancomycinresistant Enterococcus faecium bacteraemia, although the effect against this organism is bacteristatic.
9 Polypeptide antibiotics The polypeptide antibiotics comprise a rather diverse group. They include: 1 Bacitracin, with activity against Gram-positive but not Gram-negative bacteria (except Gramnegative cocci); 2 Polymyxins, which are active against many types of Gram-negative bacteria (including Ps. aeruginosa but excluding cocci, Serratia marcescens and Proteus spp.) but not Gram-positive organisms; and 3 Antitubercular antibiotics, capreomycin and viomycin. Because of its highly toxic nature when administered parenterally, bacitracin is normally restricted to external usage. 169
Chapter 10
Fig. 10.14 Lincosamides: A, lincomycin;
B, clindamycin.
The antibacterial activity of five members (A to E) of the polymyxin group is of a similar nature. However, they are all nephrotoxic, although this effect is much reduced with polymyxins B and E (colistin). Colistin sulphomethate sodium is the form of colistin used for parenteral administration. Sulphomyxin sodium, a mixture of sulphomethylated polymyxin B and sodium bisulphite, has the action and uses of polymyxin B sulphate, but is less toxic. Capreomycin and viomycin show activity against M. tuberculosis and may be regarded as being second-line antitubercular drugs.
10 Glycopeptide antibiotics Two important glycopeptide antibiotics are vancomycin and teicoplanin. 10.1 Vancomycin Vancomycin is an antibiotic isolated from Strep. orientalis and has an empirical formula of C66H75Cl2N9O4 (mol. wt 1448); it has a complex 170
tricyclic glycopeptide structure. Modern chromatographically purified vancomycin gives rise to fewer side-effects than the antibiotic produced in the 1950s. Vancomycin is active against most Grampositive bacteria, including methicillin-resistant strains of Staph. aureus and Staph. epidermidis, Enterococcus faecalis, Clostridium difficile and certain Gram-negative cocci. Gram-negative bacilli, mycobacteria and fungi are not susceptible. Vancomycin-resistant enterococci are now posing a clinical problem in hospitals, however. Vancomycin is bactericidal to most susceptible bacteria at concentrations near its minimum inhibitory concentration (MIC) and is an inhibitor of bacterial cell wall peptidoglycan synthesis, although at a site different from that of b-lactam antibiotics (Chapter 12). Employed as the hydrochloride and administered by dilute intravenous injection, vancomycin is indicated in potentially life-threatening infections that cannot be treated with other effective, less toxic, antibiotics. Oral vancomycin is the drug of choice in the treatment of antibiotic-induced pseudomembranous colitis associated with the administration
Types of antibiotics and synthetic antimicrobial agents A N(CH3)2
CH3 N
H2C CH2
N
OO H O H
H
H H
S H
N
N
H
N O
CH2
CH3
CH3 NH O
O
O H
H
O
NH
H
H
OH
C O N B O H OH N
H
H
H3C
CH3
O
O
O
H H3C
N CH CH3
O H
N
O
O
S O Fig. 10.15 Streptogramins: A,
quinupristin; B, dalfopristin.
of antibiotics such as clindamycin and lincomycin (section 7). 10.2 Teicoplanin Teicoplanin is a naturally occurring complex of five closely related tetracyclic molecules. Its mode of action and spectrum of activity are essentially similar to vancomycin, although it might be less active against some strains of coagulase-negative staphylococci. Teicoplanin can be administered by intramuscular injection.
H CH2 CH2
N(C2H5)2
11 Miscellaneous antibacterial antibiotics Antibiotics described here (Fig. 10.16) are those which cannot logically be considered in any of the other groups above. 11.1 Chloramphenicol Originally produced from a Streptomyces, chloramphenicol (Fig. 10.16A) has since been totally synthesized. It has a broad spectrum of activity, but exerts a bacteristatic effect. It has antirickettsial ac171
Chapter 10
Fig. 10.16 Miscellaneous antibiotics: A, chloramphenicol; B, fusidic acid; C, mupirocin.
tivity and is inhibitory to the larger viruses. Unfortunately, aplastic anaemia, which is dose-related, may result from treatment in a proportion of patients. It should thus not be given for minor infections and its usage should be restricted to cases where no effective alternative exists, e.g. typhoid fever resistant to other antibiotics (see Chapter 14). Some bacteria (see Chapter 13) can produce an enzyme, chloramphenicol acetyltransferase, that acetylates the hydroxyl groups in the side-chain of the antibiotic to produce, initially, 3acetoxychloramphenicol and, finally, 1,3diacetoxychloramphenicol, which lacks antibacterial activity. The design of fluorinated derivatives of chloramphenicol that are not acetylated by this enzyme could be a significant finding, although toxicity may be a problem. The antibiotic is administered orally as the palmitate, which is tasteless; this is hydrolysed to chloramphenicol in the gastrointestinal tract. The highly water-soluble chloramphenicol sodium succinate is used in the parenteral formulation, and thus acts as a pro-drug. A semisynthetic derivative of chloramphenicol, thiamphenicol, is claimed to be less toxic than chloramphenicol.
172
11.2 Fusidic acid Employed as a sodium salt, fusidic acid (Fig. 10.16B) is active against many types of Grampositive bacteria, especially staphylococci, although streptococci are relatively resistant. It is employed in the treatment of staphylococcal infections, including strains resistant to other antibiotics. However, bacterial resistance may occur in vitro and in vivo. 11.3 Mupirocin (pseudomonic acid A) Mupirocin (Fig. 10.16C) is the main fermentation product obtained from Ps. fluorescens. Other pseudomonic acids (B, C and D) are also produced. Mupirocin is active predominantly against staphylococci and most streptococci, but Enterococcus faecalis and Gram-negative bacilli are resistant. There is also evidence of plasmid-mediated mupirocin resistance in some clinical isolates of Staph. aureus. Mupirocin is employed topically in eradicating nasal and skin carriage of staphylococci, including methicillin-resistant Staph. aureus (MRSA) colonization.
Types of antibiotics and synthetic antimicrobial agents
12 Antifungal antibiotics In contrast to the wide range of antibacterial antibiotics, there are very few antifungal antibiotics that can be used systemically. Lack of toxicity is, as always, of paramount importance, but the differences in structure of, and some biosynthetic processes in, fungal cells (Chapter 4) mean that antibacterial antibiotics are usually inactive against fungi. Fungal infections are normally less virulent in nature than are bacterial or viral infections but may, nevertheless, pose a problem in individuals with a depressed immune system, e.g. AIDS sufferers. 12.1 Griseofulvin This is a metabolic by-product of Penicillium griseofulvum. Griseofulvin (Fig. 10.17A) was first isolated in 1939, but it was not until 1958 that its antifungal activity was discovered. It is active against the dermatophytic fungi, i.e. those such as Trichophyton causing ringworm. It is ineffective against Candida albicans, the causative agent of oral thrush and intestinal candidiasis, and against bacteria, and there is thus no disturbance of the normal bacterial flora of the gut. Griseofulvin is administered orally in the form of tablets. It is not totally absorbed when given orally, and one method of increasing absorption is to reduce the particle size of the drug. Griseofulvin is deposited in the deeper layers of the skin and in hair keratin, and is therefore employed in chemotherapy of fungal infections of these areas caused by susceptible organisms. 12.2 Polyenes Polyene antibiotics are characterized by possessing a large ring containing a lactone group and a hydrophobic region consisting of a sequence of four to seven conjugated double bonds. The most important polyenes are nystatin and amphotericin B (Fig. 10.17B and C, respectively). Nystatin has a specific action on C. albicans and is of no value in the treatment of any other type of infection. It is poorly absorbed from the gastrointestinal tract; even after very large doses, the blood
level is insignificant. It is administered orally in the treatment of oral thrush and intestinal candidiasis infections. Amphotericin B is particularly effective against systemic infections caused by C. albicans and Cryptococcus neoformans. It is poorly absorbed from the gastrointestinal tract and is thus usually administered by intravenous injection under strict medical supervision. Amphotericin B methyl ester (Fig. 10.17C) is water-soluble, unlike amphotericin B itself, and can be administered intravenously as a solution. The two forms have equal antifungal activity but higher peak serum levels are obtained with the ester. Although the ester is claimed to be less toxic, neurological effects have been observed. An ascorbate salt has recently been described which is water-soluble, of similar activity and less toxic. Lipid-based and liposomal formulations of amphotericin are also available which exhibit lower toxicity than conventional aqueous formulations; they may therefore be given in higher doses.
13 Synthetic antimicrobial agents 13.1 Sulphonamides Sulphonamides were introduced by Domagk in 1935. It had been shown that a red azo dye, prontosil (Fig. 10.18B), had a curative effect on mice infected with b-haemolytic streptococci; it was subsequently found that in vivo, prontosil was converted into sulphanilamide. Chemical modifications of the nucleus of sulphanilamide (see Fig. 10.18A) gave compounds with higher antibacterial activity, although this was often accompanied by greater toxicity. In general, it may be stated that the sulphonamides have a broadly similar antibacterial activity but differ widely in pharmacological actions. Bacteria that are almost always sensitive to the sulphonamides include Strep. pneumoniae, bhaemolytic streptococci, Escherichia coli and Proteus mirabilis; those almost always resistant include Enterococcus faecalis, Ps. aeruginosa, indolepositive Proteus and Klebsiella; whereas bacteria showing a marked variation in response include Staph. aureus, gonococci, H. influenzae and hospital strains of E. coli and Pr. mirabilis. 173
Chapter 10
Fig. 10.17 Antifungal antibiotics: A, griseofulvin; B, nystatin; C, amphotericin (R = H) and its methyl ester (R = CH3).
The sulphonamides show considerable variation in the extent of their absorption into the bloodstream. For example, sulphadimidine and sulphadiazine are rapidly absorbed, whereas succinylsulphathiazole and phthalylsulphathiazole are poorly absorbed and are excreted unchanged in the faeces. From a clinical point of view, the sulphonamides were extremely useful for the treatment of uncomplicated urinary tract infections caused by E. coli in domiciliary practice, although their value has diminished in recent years due to resistance develop174
ment. They have also been employed in treating meningococcal meningitis (again, the number of sulphonamide-resistant meningococcal strains is a current problem) and superficial eye infections. 13.2 Diaminopyrimidine derivatives Small-molecule diaminopyrimidine derivatives were shown in 1948 to have an antifolate action. Subsequently, compounds were developed that were highly active against human cells (e.g. the use of methotrexate as an anticancer agent), protozoa
Types of antibiotics and synthetic antimicrobial agents
Fig. 10.18 A, Some sulphonamides; B, prontosil rubrum; C, unsubstituted diaminobenzylpyrimidines; D, trimethoprim;
E, tetroxoprim; F, dapsone.
(e.g. the use of pyrimethamine in malaria) or bacteria (e.g. trimethoprim: Fig. 10.18D). Unsubstituted diaminobenzylpyrimidines (Fig. 10.18C) bind poorly to bacterial dihydrofolate reductase (DHFR). The introduction of one, two or especially three methoxy groups (as in trimethoprim) produces a highly selective antibacterial agent. A more recent antibacterial addition is tetroxoprim (2,4-diamino-5-(3¢,5¢-dimethoxy-4¢methoxyethoxybenzyl) pyrimidine; Fig. 10.18E) which retains methoxy groups at R1 and R3 and has a methoxyethoxy group at R2. Trimethoprim and tetroxoprim have a broad spectrum of activity but
resistance can arise from a non-susceptible target site, i.e. an altered DHFR (see Chapter 13). 13.3 Co-trimoxazole Co-trimoxazole is a mixture of sulphamethoxazole (five parts) and trimethoprim (one part). The reason for using this combination is based upon the in vitro finding that there is a ‘sequential blockade’ of folic acid synthesis, in which the sulphonamide is a competitive inhibitor of dihydropteroate synthetase and trimethoprim inhibits DHFR (see Chapter 12). The optimum ratio of the two components may not 175
Chapter 10
be achieved in vivo and arguments continue as to the clinical value of co-trimoxazole, with many advocating the use of trimethoprim alone. Cotrimoxazole is the agent of choice in treating pneumonias caused by Pneumocystis carinii, a yeast (although it had been classified as a protozoan). Pneumocystis carinii is a common cause of pneumonia in patients receiving immunosuppressive therapy and in those with AIDS. 13.4 Dapsone Dapsone (diaminodiphenylsulphone; Fig. 10.18F) is used specifically in the treatment of leprosy. However, because resistance to dapsone is unfortunately now well known, it is recommended that dapsone be used in conjunction with rifampicin and clofazimine. 13.5 Antitubercular drugs The three standard drugs used in the treatment of tuberculosis were streptomycin (considered above), p-aminosalicylic acid (PAS) and isoniazid (isonicotinylhydrazide, INH; synonym, isonicotinic acid hydrazine, INAH). The tubercle bacillus rapidly becomes resistant to streptomycin, and the role of PAS was mainly that of preventing this development of resistance. The current approach is to treat tuberculosis in two phases: an initial phase where a combination of three drugs is used to reduce the bacterial level as rapidly as possible, and a continuation phase in which a combination of two drugs is employed. Front-line drugs are isoniazid, rifampicin, streptomycin and ethambutol. Pyrazinamide, which has good meningeal penetration, and is thus particularly useful in tubercular meningitis, may be used in the initial phase to produce a highly bactericidal response. Isoniazid has no significant effect against organisms other than mycobacteria. It is given orally. Cross-resistance between it, streptomycin and rifampicin has not been found to occur. When bacterial resistance to these primary agents exists or develops, treatment with the secondary antitubercular drugs has to be considered. The latter group comprises capreomycin, cycloser176
ine, some of the newer macrolides (azithromycin, clarithromycin), 4-quinolones (e.g. ciprofloxacin, ofloxacin) and prothionamide (no longer marketed in the UK). Prothionamide, pyrazinamide and ethionamide are, like isoniazid, derivatives of isonicotinic acid. The British National Formulary no longer lists ethionamide as being a suitable antitubercular drug. Chemical structures of the above, and of thiacetazone (not used nowadays because of its side-effects) are presented in Fig. 10.19. There has, unfortunately, been a global resurgence of tuberculosis in recent years. Multiple drugresistant M. tuberculosis (MDRTB) strains have been isolated in which resistance has been acquired to many drugs used in the treatment of this disease. 13.6 Nitrofuran compounds The nitrofuran group of drugs (Fig. 10.20) is based on the finding over 40 years ago that a nitro group in the 5 position of 2-substituted furans endowed these compounds with antibacterial activity. Many hundreds of such compounds have been synthesized, but only a few are in current therapeutic use. In the most important nitrofurans, an azomethine group, —CH=N—, is attached at C-2 and a nitro group at C-5. Less important nitrofurans have a vinyl group, —CH=CH—, at C-2. Biological activity is lost if: 1 the nitro ring is reduced; 2 the —CH=N— linkage undergoes hydrolytic decomposition; or 3 the —CH=CH—- linkage is oxidized. The nitrofurans show antibacterial activity against a wide spectrum of microorganisms, but furaltadone has now been withdrawn from use because of its toxicity. Furazolidone has a very high activity against most members of the Enterobacteriaceae, and has been used in the treatment of diarrhoea and gastrointestinal disturbances of bacterial origin. Nitrofurantoin is used in the treatment of urinary tract infections; antibacterial levels are not reached in the blood and the drug is concentrated in the urine. It is most active at acid pH. Nitrofurazone is used mainly as a topical agent in the treatment of burns and wounds and also in certain types of ear infections. The nitrofurans are believed to be mutagenic.
Types of antibiotics and synthetic antimicrobial agents
Fig. 10.19 Antitubercular compounds
(see text also for details of antibiotics): A, PAS; B, isoniazid; c, ethionamide; D, pyrazinamide; E, prothionamide; F, thiacetazone; G, ethambutol.
Fig. 10.20 A, Furan; B, 5-nitrofurfural;
C–F, nitrofuran drugs: respectively C, nitrofurazone, D, nitrofurantoin, E, furazolidone and F, furaltadone.
177
Chapter 10
Fig. 10.21 Quinolone and antibacterial 4-quinolones. Note that the newer fluoroquine derivatives (e.g. norfloxacin,
ciprofloxacin, ofloxacin) have a 6-fluoro and a 7-piperazino substituent. Drugs marked with an asterisk are difluorinated quinolones, with a second fluorine atom at C-8.
13.7 4-Quinolone antibacterials Over 10 000 quinolone antibacterial agents have now been synthesized. Nalidixic acid is regarded as the progenitor of the new quinolones. It has been used for several years as a clinically important drug in the treatment of urinary tract infections. Since its clinical introduction, other 4-quinolone antibacterial agents have been synthesized, some of which show considerably greater antibacterial potency. Furthermore, this means that many types of bacteria not susceptible to nalidixic acid therapy may be sensitive to the newer derivatives. The most important development was the introduction of a fluorine substituent at C-6, which led to a considerable increase in potency and spectrum of activity compared with nalidixic acid. These second-generation quinolones are known as fluoroquinolones, examples of which are ciprofloxacin and norfloxacin (Fig. 10.21). Nalidixic acid is unusual in that it is active against several different types of Gram-negative bacteria, whereas Gram-positive organisms are resistant. 178
However, the newer fluoroquinolone derivatives show superior activity against Enterobacteriaceae and Ps. aeruginosa, and their spectrum also includes staphylococci but not streptococci. Extensive studies with norfloxacin have demonstrated that its broad spectrum, high urine concentration and oral administration make it a useful drug in the treatment of urinary infections. Ciprofloxacin may be used in the treatment of organisms resistant to other antibiotics; it can also be used in conjunction with a b-lactam or aminoglycoside antibiotic, e.g. when severe neutropenia is present. The third and most recently developed generation of quinolones has maintained many of the properties of the second generation; examples are lomefloxacin, sparfloxacin (both difluorinated derivatives) and temafloxacin (a trifluorinated derivative). Lomefloxacin has a sufficiently long half-life to allow once-daily dosing, but adverse photosensitivity reactions are now being recognized. Sparfloxacin retains high activity against Gramnegative bacteria but has enhanced activity against Gram-positive cocci and anaerobes. Temafloxacin
Types of antibiotics and synthetic antimicrobial agents Table 10.4 Antimicrobial imidazoles Antimicrobial or other activity Antibacterial Antiprotozoal Anthelmintic Antifungal
Examples Metronidazole, tinidazole: anaerobic bacteria only Metronidazole, tinidazole Mebendazole Clotrimazole, miconazole, econazole, ketoconazole Newer imidazoles: fluconazole, itraconazole
has, unfortunately, been withdrawn from clinical use because of unexpected severe haemolytic and nephrotoxic reactions. 13.8 Imidazole derivatives The imidazoles comprise a large and diverse group of compounds with properties encompassing antibacterial (metronidazole), antiprotozoal (metronidazole), antifungal (clotrimazole, miconazole, ketoconazole, econazole) and anthelmintic (mebendazole) activity: see Table 10.4. Metronidazole (Fig. 10.22A) inhibits the growth of pathogenic protozoa, very low concentrations being effective against the protozoa Trichomonas vaginalis, Entamoeba histolytica and Giardia lamblia. It is also used to treat bacterial vaginosis caused by Gardnerella vaginalis. Given orally, it cures 90–100% of sexually transmitted urogenital infections caused by T. vaginalis. It has also been found that metronidazole is effective against anaerobic bacteria, for example B. fragilis, and against facultative anaerobes grown under anaerobic, but not aerobic, conditions. Metronidazole is administered orally or in the form of suppositories. Other imidazole derivatives include clotrimazole (Fig. 10.22B), miconazole (Fig. 10.22C) and econazole (Fig. 10.22D), all of which possess a broad antimycotic spectrum with some antibacterial activity and are used topically. Miconazole is used topically but can also be administered by intravenous or intrathecal injection in the treatment of severe systemic or meningeal fungal infections. Newer imidazoles are (a) ketoconazole (Fig. 10.22E),
which is used orally for the treatment of systemic fungal infections (but not when there is central nervous system involvement or where the infection is life-threatening), (b) fluconazole (Fig. 10.22F), which is given orally or by intravenous infusion in the treatment of candidiasis and cryptococcal meningitis. Itraconazole is well absorbed when given orally after food. 13.9 Flucytosine Flucytosine (5-fluorocytosine; Fig. 10.22G) is a narrow-spectrum antifungal agent with greatest activity against yeasts such as Candida, Cryptococcus and Torulopsis. Evidence has been presented which shows that, once inside the fungal cell, flucytosine is deaminated to 5-fluorouracil (Fig. 10.22H). This is converted by the enzyme pyrophosphorylase to 5-fluorouridine monophosphate (FUMP), diphosphate (FUDP) and triphosphate (FUTP), which inhibits RNA synthesis; 5-fluorouracil itself has poor penetration into fungi. Candida albicans is known to convert FUMP to 5-fluorodeoxyuridine monophosphate (FdUMP), which inhibits DNA synthesis by virtue of its effect on thymidylate synthetase. Resistance can occur in vivo by reduced uptake into fungal cells of flucytosine or by decreased accumulation of FUTP and FdUMP. 13.10 Synthetic allylamines Terbinafine (Fig. 10.22I), a member of the allylamine class of antimycotics, is an inhibitor of the enzyme squalene epoxidase in fungal ergosterol biosynthesis. Terbinafine is orally active, is fungicidal and is effective against a broad range of dermatophytes and yeasts. It can also be used topically as a cream. 13.11 Synthetic thiocarbamates The synthetic thiocarbamates, of which tolnaftate (Fig. 10.22J) is an example, also inhibit squalene epoxidase. Tolnaftate inhibits this enzyme from C. albicans, but is inactive against whole cells, presumably because of its inability to penetrate the cell wall. Tolnaftate is used topically in the treatment or prophylaxis of tinea. 179
Chapter 10
Fig. 10.22 Imidazoles (A–F): A, metronidazole; B, clotrimazole; C, miconazole; D, econazole; E, ketoconazole; F, fluconazole;
G, flucytosine; H, 5-fluorouracil; I, terbinafine; J, tolnaftate.
Types of antibiotics and synthetic antimicrobial agents
13.12 Oxazolidinones The oxazolidinones are a new class of synthetic antimicrobial agents. Produced in 1987, they were found to be active in vitro against antibioticsusceptible and -resistant cocci and did not demonstrate cross-resistance with any other antibiotics. Linezolid (Fig. 10.23), a totally synthetic 3-(fluorophenyl)-2-oxalidinone, was selected for use in clinical trials. It has subsequently been introduced into clinical medicine and appears to have a useful role to play. Already, however, linezolid-resistant MRSA strains have been described. O
O
N
N
O
O
C F
N H
CH3
Fig. 10.23 Linezolid.
14 Antiviral drugs
Viruses literally ‘take over’ the machinery of an infected human cell and thus an antiviral drug must be remarkably selectively toxic if it is to inhibit the viral particle without adversely affecting the human cell. Consequently, in comparison with antibacterial agents, very few inhibitors can be considered as being safe antiviral drugs, although the situation is improving. Possible sites of attack by antiviral agents include prevention of adsorption of a viral particle to the host cell, prevention of the intracellular penetration of the adsorbed virus, and inhibition of protein or nucleic acid synthesis (Chapter 5). Genetic information for viral reproduction resides in its nucleic acid (DNA or RNA: see Chapter 5). The viral particle (virion) does not possess enzymes necessary for its own replication; after entry into the host cell, the virus uses the enzymes already present or induces the formation of new ones. Viruses replicate by synthesis of their separate components followed by assembly. Antiviral drugs are considered below with a summary in Table 10.5. 14.1 Amantadines
Several compounds are known that are inhibitory to mammalian viruses in tissue culture, but only a few can be used in the treatment of human viral infections. The main problem in designing and developing antiviral agents is the lack of selective toxicity that is normally possessed by most compounds.
Amantadine hydrochloride (Fig. 10.24A) does not prevent adsorption but inhibits viral penetration. It has a very narrow spectrum and is used prophylactically against infection with influenza A virus; it has no prophylactic value with other types of influenza virus.
Table 10.5 Examples of antiviral drugs and their clinical uses* Group
Antiviral drug
Clinical uses
Nucleoside analogues
Idoxuridine Ribavirin (tribavirin) Zidovudine Didanosine (DDI) Zalcitabine (DDC) Acyclovir (aciclovir) Famciclovir Ganciclovir Foscarnet Amantadine Protease inhibitors
Skin including herpes labialis Severe respiratory syncytial virus bronchiolitis in infants and children AIDS treatment AIDS treatment AIDS treatment Herpes simplex and varicella zoster Cytomegalovirus infections in immunocompromised patients only Cytomegalovirus retinitis in patients with AIDS Prophylaxis: influenza A outbreak HIV HIV infection
Non-nucleoside analogues
* For further information, see the current issue of the British National Formulary.
181
Chapter 10
Fig. 10.24 A, Amantadine (used as the hydrochloride);
B, methisazone.
14.2 Methisazone Methisazone (Fig. 10.24B) inhibits DNA viruses (particularly vaccinia and variola) but not RNA viruses, and has been used in the prophylaxis of smallpox. It is now little used, especially as, according to the World Health Organization, smallpox has been eradicated. 14.3 Nucleoside analogues Various nucleoside analogues have been developed that inhibit nucleic acid synthesis. Idoxuridine (2¢-deoxy-5-iodouridine; IUdR; Fig. 10.25C) is a thymidine analogue which inhibits the utilization of thymidine (Fig. 10.25A) in the rapid synthesis of DNA that normally occurs in herpes-infected cells. Unfortunately, because of its toxicity, idoxuridine is unsuitable for systemic use and it is restricted to topical treatment of herpes-infected eyes. Other nucleoside analogues include the following: cytarabine (cytosine arabinoside; Ara-C; Fig. 10.25D) which has antineoplastic and antiviral properties and which has been employed topically to treat herpes keratitis resistant to idoxuridine; adenosine arabinoside (Ara-A; vidarabine); and ribavirin (1b-d-ribofuranosyl-1,2,4-triazole-3, carboxamide; Fig. 10.25E) which has a broad spectrum of activity, inhibiting both RNA and DNA viruses. Vidarabine, in particular, has a high degree of selectivity against viral DNA replication and is primarily active against herpesviruses and some poxviruses. It may be used systemically or topically. It is related structurally to guanosine (Fig. 10.25B). Human immunodeficiency virus (HIV) is a retrovirus, i.e. its RNA is converted in human cells by the enzyme reverse transcriptase to DNA, which is in182
corporated into the human genome and is responsible for producing new HIV particles. Zidovudine (azidothymidine, AZT; Fig. 10.25F) is a structural analogue of thymidine (Fig. 10.25A) and is used to treat AIDS patients. Zidovudine is converted in both infected and uninfected cells to the mono-, diand eventually tri-phosphate derivatives. Zidovudine triphosphate, the active form, is a potent inhibitor of HIV replication, being mistaken for thymidine by reverse transcriptase. Premature chain termination of viral DNA ensues. However, AZT is relatively toxic because, as pointed out above, it is converted to the triphosphate by cellular enzymes and is thus also activated in uninfected cells. 2¢3¢-Dideoxycytidine (DDC, zalcitabine), a nucleoside analogue that also inhibits reverse transcriptase, is more active than zidovudine in vitro, and (unlike zidovudine) does not suppress erythropoiesis. DDC is not without toxicity, however, and a severe peripheral neurotoxicity, which is doserelated, has been reported. The chemical structures of DDC and of another analogue with similar properties, 2¢3¢-dideoxyinosine (DDI, didanosine), are presented in Fig. 10.25 (G, H, respectively). Didanosine is a nucleoside reverse transcriptase inhibitor structurally related to inosine. It is converted intracellularly to dideoxyadenosine triphosphate. It acts against retroviruses, including HIV. Aciclovir (acycloguanosine, Fig. 10.25I) is a novel type of nucleoside analogue that becomes activated only in herpes-infected host cells by a herpes-specific enzyme, thymidine kinase. This enzyme initiates conversion of aciclovir initially to a monophosphate and then to the antiviral triphosphate which inhibits viral DNA polymerase. The host cell polymerase is not inhibited to the same extent, and the antiviral triphosphate is not produced in uninfected cells. Ganciclovir (Fig. 10.25J) is up to 100 times more active than aciclovir against human cytomegalovirus (CMV) but is also much more toxic; it is reserved for the treatment of severe CMV in immunocompromised patients. Famciclovir is similar, being a pro-drug of penciclovir (an antiviral with similar activity to aciclovir) and valociclovir is a pro-drug ester of aciclovir.
Types of antibiotics and synthetic antimicrobial agents
Fig. 10.25 Thymidine (A), guanosine (B) and some nucleoside analogues (C–J). C, idoxuridine; D, cytarabine; E, ribavirin;
F, zidovudine (AZT); G, dideoxycytidine (DDC); H, dideoxyinosine (DDI); I, acyclovir; J, ganciclovir.
14.4 Non-nucleoside compounds 14.4.1 Phosphonoacetic acid and sodium phosphonoformate Apart from the amantadines (section 14.1) and methisazone (section 14.2), various non-nucleoside drugs have shown antiviral activity. Two simple molecules with potent activity are phosphonoacetic
acid (Fig. 10.26A) and sodium phosphonoformate (foscarnet, Fig. 10.26B). Phosphonoacetic acid has a high specificity for herpes simplex DNA synthesis, and has been shown to be non-mutagenic in experimental animals, but is highly toxic. Foscarnet inhibits herpes DNA polymerase, is non-toxic when applied to the skin and is a potentially useful agent in treating herpes simplex labialis (cold sores). It is 183
Chapter 10 A
O
HO
P
B
O –O
CH2COOH
O
P
C
O–
OH
3 Na+ Fig. 10.26 A, Phosphonoacetic acid; B, sodium
O–
phosphonoformate (foscarnet).
B
A
H N N O H2NOC
O
O
NHtBu
N O
N H
Ph O
OH
H N
S
N
H
O
OH
N H
CH3
H N
S
H N O
Ph
H D
C OH
N N BuHtN
O
Ph H N O
OH
H2SO4
O HO
S
O
N H
N
NHtBu CH3SO3H H
OH H Fig. 10.27 HIV protease inhibitors: A, saquinavir; B, ritonavir; C, inidavir; D, nelfinavir.
used for cytomegalovirus retinitis in patients with AIDS in whom ganciclovir is inappropriate. Tetrahydroimidazobenzodiazepinone (TIBO) compounds have shown excellent activity in vitro against HIV reverse transcriptase in HIV type 1 (HIV-1) but not HIV-2 or other retroviruses. 14.4.2 Protease inhibitors New antiviral agents have been based on virus-specific targets, especially the viral enzymes that are involved in the viral life cycle, i.e. responsible for the production of infectious viral particles. Research has focused on virus-encoded proteases that fulfil this role. Thus, specific inhibitors of HIV, herpesvirus and hepatitis C virus proteases are now known. Examples of clinically approved HIV pro184
tease inhibitors are saquinavir, ritonavir, indinavir and nelfinavir (Fig. 10.27A–D). 14.5 Interferons Interferon is a low molecular weight protein, produced by virus-infected cells, that itself induces the formation of a second protein inhibiting the transcription of viral mRNA. Interferon is produced by the host cell in response to the virus particle, the viral nucleic acid and non-viral agents, including synthetic polynucleotides such as polyinosinic acid and polycytidylic acid (poly I:C). There are two types of interferon. Type I interferons. These are acid-stable and comprise two major classes, leucocyte interferon (LeIFN, IFN-a) released by stimulated leucocytes,
Types of antibiotics and synthetic antimicrobial agents
and fibroblast interferon (F-IFN, IFN-b) released by stimulated fibroblasts. Type II interferons. These are acid-labile and are also known as ‘immune’ (IFN-g) interferons because they are produced by T lymphocytes (see Chapter 8) in the cellular immune system in response to specific antigens. Type I interferons induce a virus-resistant state in human cells, whereas type II are more active in inhibiting growth of tumour cells. Disappointingly low yields of F-IFN and Le-IFN are achieved from eukaryotic cells but recombinant DNA technology has been employed to produce interferon in prokaryotic cells (bacteria). This aspect is considered in more detail in Chapter 24.
sidered to be much fewer than originally thought. There is also the problem of a chemical or physical incompatibility between two drugs. Examples where combinations have an important role to play in antibacterial chemotherapy were provided earlier (sections 2.3 and 2.9) in which a b-lactamase inhibitor and an appropriate b-lactamase-labile penicillin form a single pharmaceutical product, and in section 8 (streptogramins). Further comment on drug combinations can be found in Chapter 14. It must also be noted that a combination of two blactams does not necessarily produce a synergistic effect. Some antibiotics are excellent inducers of blactamase, and consequently a reduced response (antagonism) may be produced.
15 Drug combinations
16 Further reading
A combination of two antibacterial agents may produce the following responses. 1 Synergism, where the joint effect is greater than the sum of the effects of each drug acting alone. 2 Additive effect, in which the combined effect is equal to the arithmetic sum of the effects of the two individual agents. 3 Antagonism (interference), in which there is a lesser effect of the mixture than that of the more potent drug action alone. There are four possible justifications as to the use of antibacterial agents in combination. 1 The concept of clinical synergism, which may be extremely difficult to demonstrate convincingly. Even with trimethoprim plus sulphamethoxazole, where true synergism occurs in vitro, the optimum ratio of the two components may not always be present in vivo, i.e. at the site of infection in a particular tissue. 2 A wider spectrum of cover may be obtained, which may be (a) desirable as an emergency measure in life-threatening situations; or (b) of use in treating mixed infections. 3 The emergence of resistant organisms may be prevented. A classical example here occurs in combined antitubercular therapy (see earlier). 4 A possible reduction in dosage of a toxic drug may be achieved. Indications for combined therapy are now con-
Axelsen, P. H. (2002) Essentials of Antimicrobial Pharmacology. Humana Press, Totowa, NJ. Bean, B. (1992) Antiviral therapy: current concepts and practices. Clin Microbiol Rev, 5, 146–182. Bowden, K., Hants, N.V. & Watson, C.A. (1993) Structureactivity relationships of dihydrofolate reductase inhibitors. J Chemother, 5, 377–388. Bugg, C.E., Carson, W.M. & Montgomery, J.A. (1993) Drugs by design. Sci Am, 269, 92–98. British National Formulary. British Medical Association & Pharmaceutical Press, London. [The chapter on drugs used in the treatment of infections is a particularly useful section. New editions of the BNF appear at regular intervals] Brown, A.G. (1981) New naturally occurring b-lactam antibiotics and related compounds. J Antimicrob Chemother, 7, 15–48. Chopra, I. (1998) Research and development of antibacterial agents. Curr Opinion Microbiol, 1, 855–869. Chopra, I., Hawkey, P.M. & Hinton, M. (1992) Tetracyclines, molecular and clinical aspects. J Antimicrob Chemother, 29, 245–277. Cowan, M.M. (1999) Plant products as antimicrobial agents. Clin Microbiol Rev, 12, 564–582. De Clerq, E. (1997) In search of a selective antiviral chemotherapy. Clin Microbiol Rev, 10, 674–693. Finch, R.G., Greenwood, D., Norrby, R. & Whitley, R. (2002) Antibiotic and Chemotherapy, 8th edn. Churchill Livingstone, London and Edinburgh. Hamilton-Miller, J.M.T. (1991) From foreign pharmacopoeias: ‘new’ antibiotics from old? J Antimicrob Chemother, 27, 702–705. Hooper, D.C. & Wolfson, J.S. (1993) Quinolone Antimicrobial Agents, 2nd edn. American Society for Microbiology, Washington.
185
Chapter 10 Hunter, P.A., Darby, G.K. & Russell, N.J. (1995) Fifty Years of Antimicrobials: Past Perspectives and Future Trends. 53rd Symposium of the Society for General Microbiology. Cambridge University Press, Cambridge. Kuntz, I.D. (1992) Structure-based strategies for drug design and discovery. Science, 257, 1079–1082. Patick, A.K. & Potts, K.E. (1998) Protease inhibitors as antiviral agents. Clin Microbiol Rev, 11, 614–627. Power, E.G.M. & Russell, A.D. (1998) Design of antimicrobial chemotherapeutic agents. In: Smith, H.J., ed. Introduction to Principles of Drug Design, 3rd edn. Wright, Bristol.
186
Reeves, D.S. & Howard, A.J. (1991) New macrolides — the respiratory antibiotics for the 1990s. J Hosp Infect, 19 (Suppl. A). Russell, A.D. & Chopra, I. (1996) Understanding Antibacteral Action and Resistance, 2nd edn. Ellis Horwood, Chichester. Sammes, P.G. (1997–1982) Topics in Antibiotic Chemistry, vols 1–5. Ellis Horwood, Chichester. Shanson, D.C. (1999) Microbiology in Clinical Practice, 3rd edn. Wright, London.
Chapter 11
Laboratory evaluation of antimicrobial agents JMB Smith 1 Introduction 1.1 Definitions 2 Factors affecting the antimicrobial activity of disinfectants 2.1 Innate (natural) resistance of microorganisms 2.2 Microbial density 2.3 Disinfectant concentration and exposure time 2.4 Physical and chemical factors 2.4.1 Temperature 2.4.2 pH 2.4.3 Divalent cations 2.5 Presence of extraneous organic material 3 Evaluation of liquid disinfectants 3.1 General 3.2 Antibacterial disinfectant efficacy tests 3.2.1 Suspension tests 3.2.2 In-use and simulated use tests 3.2.3 Problematic bacteria 3.3 Other microbe disinfectant tests
1 Introduction As the range of microorganisms acquiring resistance to the presently available antimicrobial agents continues to increase, evaluation of the potential antimicrobial action and nature of the inhibitory and lethal effects of established and novel therapeutic agents and biocides has re-surfaced as important laboratory procedures. A linkage between biocide usage and antibiotic resistance potentially exists. In addition, the emergence of new infectious agents (e.g. prions) and the escalating occurrence of significant bloodborne viruses (e.g. human immunodeficiency viruses, hepatitis B and C) which may readily contaminate medical instruments or the environment has jolted into reality the need for suitable and proven liquid disinfecting and sterilizing agents. Clearly the availability and efficacy of such chemical agents must be backed by appropriate laboratory data. The events of September
4 5 6 7 8
9
3.3.1 Antifungal (fungicidal) tests 3.3.2 Antiviral (virucidal) tests 3.3.3 Prion disinfection tests Evaluation of solid disinfectants Evaluation of air disinfectants Evaluation of preservatives Rapid evaluation procedures Evaluation of potential chemotherapeutic antimicrobials 8.1 Tests for bacteriostatic activity 8.1.1 Disc tests 8.1.2 Dilution tests 8.1.3 E-tests 8.1.4 Problematic bacteria 8.2 Tests for bactericidal activity 8.3 Tests for fungistatic and fungicidal activity 8.4 Evaluation of possible synergistic antimicrobial combinations 8.4.1 Kinetic kill curves Further reading
11, 2001, in New York and the subsequent threat of bioterrorism worldwide, together with the continuing problems of legionellosis and contaminated airconditioning units, has also renewed interest and debate into the value of biocides in the environmental control of microorganisms and microbial diseases. Tests for evaluating candidate antimicrobial agents to be used in human and animal medicine as well as environmental biocides are now significant laboratory considerations. 1.1 Definitions Key terms such as disinfection, sterilization and preservation are defined in Chapter 17 but other terms are often employed to describe the antimicrobial activity of agents. Hence, terms such as biocidal, bactericidal, virucidal and fungicidal describe a killing activity, whereas bacteriostatic and fungistatic refer to inhibition of growth of the organism 187
Chapter 11
100
Viable cells (log)
Normal growth
Effect of a static agent
Effect of a cidal agent
Log survival (%)
10
1
0.1
x Time (hours) Fig. 11.1 Effect on the subsequent growth pattern of
inhibitory (static D) or cidal (䊐) agents added at time X (the normal growth pattern is indicated by the 䊉 line).
0.01 0
1
10
100
Antimicrobial concentration (mg/L) Fig. 11.2 Survival of Enterococcus faecalis exposed to a
(Fig. 11.1). It must be remembered, however, that some microorganisms that appear to be dead and non-cultivable may be revived by appropriate methods, and that organisms incapable of multiplication may retain some enzymatic activity. When evaluating the activity of antibacterial agents the terms minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are commonly used. MIC refers to the minimum concentration of an antimicrobial agent that inhibits growth of the microorganism under test and is generally recorded in mg/L or mg/ml. With most cidal antimicrobials, the MIC and MBC are often near or equal in value, although with essentially static agents (e.g. tetracycline), the lowest concentration required to kill the microorganism (i.e. the MBC) is invariably many times the MIC and clinically unachievable without damage to the human host. Similar cidal terms can be applied to studies involving other microbes, e.g. minimum fungicidal concentration (MFC). ‘Tolerance’ is a term that implies the ability of some bacterial strains to survive, but not grow, at levels of antimicrobial agent that should normally be cidal. This applies particularly to systems employing the cell wall active b-lactams and glycopep188
fluoroquinolone for 4 h at 37°C. Three initial bacterial concentrations were studied, 107 cfu/ml (䊐); 106 cfu/ml (D) and 105 cfu/ml (䊊). This clearly demonstrates a paradoxical effect (increasing antimicrobial concentrations past a critical level reveal decreased killing), and the effects of increased inoculum densities on subsequent killing (courtesy of Dr Z. Hashmi).
tides, and to Gram-positive bacteria such as streptococci. Normally, MIC and MBC levels in such tests should be similar (i.e. within one or two doubling dilutions); if the MIC/MBC ratio is 32 or greater, the term tolerance is used. Tolerance may in some way be related to the Eagle phenomenon (paradoxical effect), where increasing concentrations of antimicrobial result in less killing rather than the expected increase in cidal activity (see Fig. 11.2).
2 Factors affecting the antimicrobial activity of disinfectants Laboratory tests for the evaluation of chemicals with potential antimicrobial activity must be scientifically based and ensure that the agent is safe and
Laboratory evaluation of antimicrobial agents
Log survival (%)
100
Control
10
Amoebae grown cells
1 Broth grown cells
increase in resistance to disinfectants and biocides (Fig. 11.3). Therefore, when evaluating new disinfectants, a suitable range of microorganisms and environmental conditions must be included in tests. The European suspension test (prEN 12054) for hospital-related studies includes Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Enterococcus hirae.
.1
2.2 Microbial density .01 0 1 2 4 6 Time (hours) Fig. 11.3 Survival of stationary phase broth cultures of
Legionella pneumophila and amoebae-grown L. pneumophila after exposure to 32 mg/L benzisothiazolone (Proxel) at 35°C in Ringer’s solution. Adapted from Barker et al. (1992), Appl Environ Microbiol, 58, 2420–2425, with permission.
effective for its intended use. As shown by Krönig and Paul in the late 1890s, bacteria exposed to a cidal agent do not die simultaneously but in an orderly sequence, and attempts at applying the kinetics of pure chemical reactions to microbe/ disinfectant interactions are often unrewarding. However, the antimicrobial/microorganism interaction is influenced by a variety of factors that may be related to natural (intrinsic) features of the microorganism involved and the chemical and physical environment in which tests are carried out. More expansive accounts concerning the dynamics of disinfection, relating largely to Chick’s pioneering work with phenol in the early 20th century, can be readily accessed (see Further reading section). 2.1 Innate (natural) resistance of microorganisms Microorganisms vary tremendously in their susceptibility to chemical disinfectants. Prions, bacterial endospores and mycobacteria possess the most innate resistance, while many vegetative bacteria and some viruses appear highly susceptible (see Chapter 17). In addition, microorganisms adhering to surfaces as biofilms or present within other cells (e.g. legionellae within amoebae), may reveal a marked
As many disinfectants require adsorption to the microbial cell surface prior to killing, dense cell populations may sequester all the available disinfectant before all cells are affected. Thus, from a practical point of view, the larger the number of microorganisms present, the longer it takes a disinfectant to complete killing of all cells. For instance, using identical test conditions, it has been shown that 10 spores of the anthrax bacillus (Bacillus anthracis) were destroyed in 30 minutes, while it took 3 hours to kill 100 000 (105) spores. The implications of washing and cleaning of objects (which removes most of the microorganisms) before disinfection, thus becomes obvious. However, when evaluating disinfectants in the laboratory, it must be remembered that unlike sterilization, kill curves with disinfectants may not be linear and the rate of killing may decrease at lower cell numbers (Fig. 11.3). Hence a 3-log killing may be more rapidly achieved with 108 than 104 cells. Cell numbers must, therefore, be standardized in disinfectant efficacy (suspension) tests and agreement reached on the degree of killing required over a stipulated time interval (see Table 11.1). Table 11.1 Methods of recording viable cells remaining after exposure of an initial population of 1 000 000 (106) CFU to a cidal agent Viable count remaining (CFU) 100 000 (105
)
10 000 (104) 1000 (103) 100 (102) 10 (101)
Log survival (%)
Log killing
% killing
10 1 0.1 0.01 0.001
1-log 2-log 3-log 4-log 5-log
90 99 99.9 99.99 99.999
189
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2.3 Disinfectant concentration and exposure time The effects of concentration or dilution of the active ingredient on the activity of a disinfectant are of major importance. Apart from iodophors, the more concentrated a disinfectant, the greater its efficacy, and the shorter the time necessary to destroy the microorganism, i.e. there is an exponential relationship between potency and concentration. Therefore, a graph plotting the log10 of a death time (i.e. the time required to kill a standard inoculum) against the log10 of the concentration is usually a straight line, the slope of which is the concentration exponent (h). Expressed as an equation:
h=
(Log death time at concentration C2 ) -(Log death time at concentration C1 ) Log C1 - C2
Thus h can be obtained from experimental data either graphically or by substitution in the above equation (see Table 11.2).
Table 11.2 Concentration exponents, h, for some disinfectant substances Antimicrobial agent
h
Hydrogen peroxide Silver nitrate Mercurials Iodine Crystal violet Chlorhexidine Formaldehyde QACs Acridines Formaldehyde donors Bronopol Polymeric biguanides Parabens Sorbic acid Potassium laurate Benzyl alcohol Aliphatic alcohols Glycolmonophenyl ethers Glycolmonoalkyl ethers Phenolic agents
0.5 0.9–1.0 0.03–3.0 0.9 0.9 2 1 0.8–2.5 0.7–1.9 0.8–0.9 0.7 1.5–1.6 2.5 2.6–3.2 2.3 2.6–4.6 6.0–12.7 5.8–6.4 6.4–15.9 4.0–9.9
QAC, quaternary ammonium compound.
190
It is clear that dilution does not affect the cidal attributes of all disinfectants in a similar manner. Mercuric chloride with a concentration exponent of 1 will be reduced by the power of 1 on dilution, and a threefold dilution means the disinfectant activity will be reduced by the value 31, or to a third of its original activity. Phenol, however, has a concentration exponent of 6, so a threefold dilution in this case will mean a decrease in activity of 36 or 729 times less active than the original. 2.4 Physical and chemical factors Known and proven influences include temperature, pH and water hardness. 2.4.1 Temperature Within limits, the cidal activity of most disinfectants increases as the temperature rises. As the temperature is increased in arithmetical progression the rate (velocity) of disinfection increases in geometrical progression. Results may be expressed quantitatively by means of a temperature coefficient, either the temperature coefficient per degree rise in temperature (q), or the coefficient per 10° rise (the Q10 value) (Hugo & Russell, 1998). As shown by Koch working with phenol and anthrax (Bacillus anthracis) spores over 120 years ago, raising the temperature of phenol from 20°C to 30°C increased the killing activity by a factor of 4 (the Q10 value). q may be calculated from the equation: q (T1 -T2 ) = t1 t2 Where t1 is the extinction time at T1°C, and t2 the extinction at T2°C (i.e. T1 + 1°C). Q10 values may be calculated easily by determining the extinction time at two temperatures differing exactly by 10°C. Then: Q10 =
Time to kill at T∞ Time to kill at (T + 10)∞
It is also possible to plot the rate of kill against the temperature. While the value for Q10 of chemical and enzymecatalysed reactions lies in a narrow range (between 2 and 3), values for disinfectants vary widely, e.g. 4
Laboratory evaluation of antimicrobial agents
for phenol, 45 for ethanol, and almost 300 for ethylene glycol monoethyl ether. Clearly, relating chemical reaction kinetics to disinfection processes is potentially dangerous. Most laboratory tests involving disinfectant-like chemicals are now standardized to 20°C, i.e. around ambient room temperatures. 2.4.2 pH Effects of pH on antimicrobial activity can be complex. As well as influencing the survival and rate of growth of the microorganism under test, changes in pH may affect the potency of the agent and its ability to combine with cell surface sites. If the agent is an acid or a base, its degree of ionization will depend on the pH. With some antimicrobials (e.g. phenols, acetic acid, benzoic acid), the non-ionized molecule is the active state and alkaline pHs which favour the formation of ions of such compounds will decrease the activity. Others (e.g. glutaraldehyde, quaternary ammonium compounds — QACs) reveal increased cidal activity as the pH rises and are best used under alkaline conditions. Adherence to cell surfaces is essential for the activity of many disinfectants; increasing the external pH renders cell surfaces more negatively charged and enhances the binding of cationic compounds such as chlorhexidine and QACs. 2.4.3 Divalent cations Divalent cations (e.g. Mg2+, Ca2+) present in hard water may also interact with the microbial cell surface and block disinfectant adsorption sites necessary for activity. On the other hand, cationic compounds may disrupt the outer membrane of Gram-negative bacteria and facilitate their own entry. 2.5 Presence of extraneous organic material It has long been known that the cidal activity of many antimicrobial agents is seriously impaired under ‘dirty’ conditions. Some of the original laboratory assays employed dried human faeces or yeast to mimic the effects of blood, pus or faeces on disinfectant activity. This is particularly true of the
halogen disinfectants (e.g. sodium hypochlorite) where the disinfectant reacts with the organic matter to form inactive complexes. It also seems that organic material may adhere to the microbial cell surface and block adsorption sites necessary for disinfectant activity. For practical purposes and to mirror potential in-use situations, disinfectants should be evaluated under both clean and dirty conditions. The latter usually includes the addition of albumin, although sheep blood or mucin have also been suggested.
3 Evaluation of liquid disinfectants 3.1 General Phenol coefficient tests were developed in the early 20th century when typhoid fever was a significant public health problem and phenolics were used to disinfect contaminated utensils and other inanimate objects. Details of such tests can be found in earlier editions of this book. However, as nonphenolic disinfectants became more widely available, tests that more closely paralleled the conditions under which disinfectants were being used (e.g. blood spills) and which included a more diverse range of microbial types (e.g. viruses, bacteria, fungi, protozoa) were developed. Evaluation of a disinfectant’s efficacy was based on its ability to kill microbes, i.e. its cidal activity, under environmental conditions mimicking as closely as possible real life situations. As an essential component of each test was a final viability assay, removal or neutralization of any residual disinfectant became a significant consideration. Around 1970, Kelsey, Sykes and Maurer developed the so-called ‘capacity-use dilution test’ which measured the ability of a disinfectant at an appropriate in-use concentration to kill successive additions of a bacterial culture. Results were reported simply as pass or fail and not a numerical cipher (coefficient). Tests employed disinfectants diluted in clean hard water and in water containing organic material (‘dirty’ conditions), with the final recovery broth containing 3% Tween 80 as a neutralizer. Such tests were applicable for use with a wide variety of disinfectants (see Kelsey & Maurer, 1974). 191
Chapter 11
However, it soon became apparent that the greatest information concerning the fate of microbes exposed to a disinfectant was obtainable by counting the number of viable cells remaining after exposure of a standard suspension of cells to the disinfectant for a given time interval — suspension tests. Viable counting is a technique used in many branches of pure and applied microbiology. Assessment of the number of viable microbes remaining after exposure allows the killing or cidal activity of the disinfectant to be expressed in a variety of ways, e.g. percentage kill (e.g. 99.999%), as a log10 reduction in numbers (e.g. 5-log killing), or by log10 survival expressed as a percentage. Examples of results are shown in Table 11.1. Unfortunately, standardization of the methodology to be employed in these efficacy tests has proved difficult, if not impossible, to obtain, as has consensus on what level of killing represents a satisfactory and/or acceptable result. It must be stressed however, that unlike tests involving chemotherapeutic agents where the major aim is to establish antimicrobial concentrations that inhibit growth (i.e. MICs), disinfectant tests require determinations of appropriate cidal levels. Levels of killing required over a given time interval tend to vary depending on the regulatory authority concerned. While a 5-log killing of bacteria (starting with 106 CFU/ml) has been suggested for suspension tests, some authorities require a 6-log killing in simulated use tests. With viruses, a 4-log killing tends to be an acceptable result, while with prions it has been recommended that a titre loss of 104 prions should be regarded as an indication of appropriate disinfection provided that there has been adequate prior cleaning. With simulated use tests, cleaning followed by appropriate disinfection should result in a prion titre loss of at least 107.
duce some form of standardization with disinfectant tests. Perhaps the most readily accessible and recent guide to the methodology of possible bactericidal, tuberculocidal, fungicidal and virucidal disinfectant efficacy tests, is that of Kampf and colleagues (2002). This publication summarizes and provides references to various EN procedures (e.g. prEN 12054). Whatever method used, however, it should generate results that are amenable to statistical analysis. 3.2.1 Suspension tests While varying to some degree in their methodology, most of the proposed procedures tend to employ a standard suspension of the microorganism in water containing albumin (dirty conditions) and appropriate dilutions of the disinfectant — so-called ‘suspension tests’. Tests are carried out at a set temperature (usually around room temperature or 20°C), and at a selected time interval samples are removed and viable counts are performed following neutralization of any disinfectant remaining in the sample. Neutralization or inactivation of residual disinfectant can be carried out by dilution, or by addition of specific agents (see Table 11.3). Using viable counts, it is possible to calculate the concentration of disinfectant required to kill 99.999% of the original suspension. Thus 10 survivors from an original population of 106 (1 000 000) cells represents a 99.999% or 5-log kill. As bacteria may initially decline in numbers in diluents devoid of additional disinfectant, results from tests incorporating disinfectant-treated cells can be compared with results from simultaneous tests involving a non-disinfectant-containing system (untreated cells). The bactericidal effect BE can then be expressed as: BE = log NC - log ND
3.2 Antibacterial disinfectant efficacy tests Various regulatory authorities in Europe (e.g. European Standard or Norm, EN; British Standards, BS; Germany, DGHM; France, AFNOR) and North America (e.g. Food and Drug Administration, FDA; Environmental Protection Authority, EPA; Association of Official Analytical Chemists, AOAC) have been associated with attempts to pro192
where NC and ND represent the final number of CFU/ml remaining in the control and disinfectant series, respectively. Unfortunately, viable count procedures carry the proviso that one colony develops from one cell or one colony-forming unit (CFU). Such techniques are, therefore, not ideal for disinfectants (e.g. QACs such as cetrimide) that promote clumping in bacter-
Laboratory evaluation of antimicrobial agents Table 11.3 Neutralizing agents for some antimicrobial agentsa
Antimicrobial agent
Neutralizing and/or inactivating agentb
Alcohols None (dilution) Alcohol-based hand gels Tween 80, saponin, histidine and lecithin Amoxycillin b-Lactamase from Bacillus cereusc Antibiotics (most) None (dilution, membrane filtrationd, resin adsorptione) Benzoic acid Dilution or Tween 80f Benzyl penicillin b-Lactamase from Bacillus cereus Bronopol Cysteine hydrochloride Chlorhexidine Lubrol W and egg lecithin or Tween 80 and lecithin (Letheen) Formaldehyde Ammonium ions Glutaraldehyde Glycine Halogens Sodium thiosulphate Hexachlorophane Tween 80 Mercurials Thioglycollic acid (-SH compounds) Phenolics Dilution or Tween 80 QACs Lubrol W and lecithin or Tween 80 and lecithin (Letheen) Sulphonamides p-Aminobenzoic acid a Other
than dilution — adapted from Hugo & Russell (1998). b D/E neutralizing media — adequate for QACs, phenols, iodine and chlorine compounds, mecurials, formaldehyde and glutaraldehyde (see Rutala, 1999). c Other appropriate enzymes can be considered — e.g. inactivating or modifying enzymes for chloramphenicol and aminoglycosides, respectively. d Filter microorganisms onto membrane, wash, transfer membrane to growth medium. e Resins for the absorption of antibiotics from fluids are available. f Tween 80 (polysorbate 80).
with an organic load and the appropriate test microorganism have been incorporated into disinfectant testing protocols. An example is the in-use test first enunciated by Maurer in 1972. It is used to determine whether the disinfectant in jars, buckets or other containers in which potentially contaminated material (e.g. lavatory brushes) has been placed contain living microorganisms, and in what numbers. A small volume of fluid is withdrawn from the in-use container, neutralized in a large volume of a suitable diluent and viable counts are performed on the resulting suspension. Two plates are involved in viable count investigations — one of which is incubated for 3 days at 32°C (rather than 37°C, as bacteria damaged by disinfectants recover more rapidly at lowered temperatures), and the other for 7 days at room temperature. Growth of one or two colonies per plate can be ignored (a disinfectant is not usually a sterilant), while 10 or more colonies would suggest poor and unsatisfactory cidal action. Simulated use tests involve deliberate contamination of instruments, inanimate surfaces, or even skin surfaces, with a microbial suspension. This may either be under clean conditions or may utilize a diluent containing organic (e.g. albumin) material — dirty condition. After being left to dry, the contaminated surface is exposed to the test disinfectant for an appropriate time interval. The microbes are then removed (e.g. by rubbing with a sterile swab), resuspended in suitable neutralizing medium, and assessed for viability as for suspension tests. New products are often compared with a known comparator compound (e.g. 1 minute application of 60% v/v 2-propanol for hand disinfection products — see EN1500) to show increased efficacy of the novel product. 3.2.3 Problematic bacteria
ial suspensions, although the latter problem may be overcome by adding non-ionic surface active agents to the diluting fluid. 3.2.2 In-use and simulated use tests Apart from suspension tests, in-use testing of used medical devices, and simulated use tests involving instruments or surfaces deliberately contaminated
Mycobacteria are hydrophobic in nature and it may be difficult to prepare homogeneous suspensions devoid of undue cell clumping. As Mycobacterium tuberculosis is very slow growing, a saprophytic rapidly growing species such as Mycobacterium terrae can be substituted in tests (as representative of M. tuberculosis). Apart from vegetative bacterial cells, spores can also be used as the inoculum in tests. In such cases, 193
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incubation of plates for the final viability determination should be continued for several days to allow for germination and growth. Compared with suspended (planktonic) cells, bacteria on surfaces as biofilms are invariably phenotypically more resistant to antimicrobial agents. With biofilms, suspension tests can be modified to involve biofilms produced on small pieces of an appropriate glass or metal substrate, or on the bottom of microtitre tray wells. After being immersed in, or exposed to the disinfectant solution for the appropriate time interval, the cells from the biofilm are removed, e.g. by sonication, and resuspended in a suitable neutralizing medium. Viable counts are then performed on the resulting planktonic cells. Some important environmental bacteria survive in nature as intracellular parasites of other microbes, e.g. Legionella pneumophila within the protozoan Acanthamoeba polyphaga. Biocide activity is significantly reduced against intracelluar legionellae (see Fig. 11.3). Disinfectant tests involving such bacteria should therefore be conducted both on planktonic bacteria and on suspensions involving amoebae-containing bacteria. With the latter, the final bacterial viable counts are performed after suitable lysis of the protozoan host. The legionellae/protozoal situation may also be complicated by the fact that the microbes often occur as biofilms. 3.3 Other microbe disinfectant tests Suspension-type efficacy tests can also be performed on other microbes, e.g. fungi, viruses, using similar techniques to that described above for bacteria, although significant differences obviously occur in parts of the tests. 3.3.1 Antifungal (fungicidal) tests Perhaps the main problem with fungi concerns the question of what to use as the inoculum. Unicellular yeasts can be treated as for bacteria, but whether to use spores (which may be more resistant than the vegetative mycelium) or pieces of hyphae with the filamentous moulds, has yet to be fully resolved. Spore suspensions (in saline containing the wetting agent Tween 80) obtained from 7-day-old cultures 194
are presently recommended. What fungus, e.g. yeast or mould, to use as the test microbe is also debatable, and will clearly vary depending on the perceived use for the disinfectant under test. Spore suspensions of at least 106 CFU/ml have been recommended. Viable counts are performed on suitable media (e.g. malt extract agar) with incubation at 20°C for 48 hours or longer. EN 1275 regulations for fungicidal activity require a minimum reduction in viability by a factor of 104 within 60 minutes; test fungi were Candida albicans and Aspergillus niger. 3.3.2 Antiviral (virucidal) tests The testing of disinfectants for virucidal activity is not easy; viruses are obligate intracellular parasites and are unable to grow in artificial culture media. They require some other system employing living cells. Suggested test viruses include rotavirus, adenovirus, poliovirus, herpes simplex viruses, human immunodeficiency virus, pox viruses and papovavirus, although extension of this list to include additional bloodborne viruses such as hepatitis B and C, and significant animal pathogens (e.g. foot and mouth disease virus) could be argued. Appropriate facilities for handling such pathogens are essential. Briefly, the virus is grown in an appropriate cell line that is then mixed with water containing an organic load and the disinfectant under test. After the appropriate time, residual viral infectivity is determined using a tissue culture/plaque assay or other system (e.g. animal host, molecular assay for some specific viral component). Such procedures are costly and time-consuming, and must be appropriately controlled to exclude factors such as disinfectant killing of the cell system or test animal. A reduction of infectivity by a factor of 104 has been regarded as evidence of acceptable virucidal activity (prEN 14476). For viruses which cannot be grown in the laboratory (e.g. hepatitis B), naturally infected cells/tissues must be used. 3.3.3 Prion disinfection tests Prions are a unique class of pathogen, devoid of an agent-specific nucleic acid (DNA or RNA). Infection is associated with the abnormal isoform of a
Laboratory evaluation of antimicrobial agents
host cellular protein called prion protein (PrPc). Prions exhibit high resistance to conventional chemical and physical decontamination methods — most studies have been done with tissue homogenates, which may offer some protective role. Hence the importance of a prior cleaning procedure before any attempted disinfection process. Current prion disinfection assays are slow, laborious and costly — they employ ‘contaminated’ test tissue homogenates (prion ‘strain’ and concentration variable, possible disinfectant inactivation), test animals which serve to assess the viability of treated tissue, with the log decrease being calculated from incubation period assays rather than from end-point titrations. While most disinfectants are inadequate for the elimination of prion infectivity, agents such as sodium hydroxide and sodium hypochlorite have been shown to be effective (Chapter 17).
4 Evaluation of solid disinfectants Solid disinfectants usually consist of a disinfectant substance diluted by an inert powder. Phenolic substances adsorbed onto kieselguhr form the basis of many disinfectant powders, while another widely used solid disinfectant is sodium dichloroisocyanurate. Other disinfectant or antiseptic powders used in medicine include acriflavine and compounds with antifungal activity such as zinc undecenoate or salicylic acid mixed with talc. These disinfectants may be evaluated by applying them to suitable test organisms growing on a solid agar medium. Discs may be cut from the agar and subcultured for enumeration of survivors. Inhibitory activity is evaluated by dusting the powders onto the surface of seeded agar plates, using the inert diluents as a control. The extent of growth is then observed following incubation.
5 Evaluation of air disinfectants
tenance of a microbe-free environment is important for many aseptic procedures. The microorganisms themselves may be contained in aerosols, or may occur as airborne particles liberated from some environmental source, e.g. decaying vegetation. While the microorganisms can be killed by suitable radiation (e.g. UV), it is often more practical to use some form of chemical vapour or aerosol to kill them. As shown by Robert Koch in 1881, the numbers of viable bacteria present in air can be assessed by simply exposing plates of solid nutrient media to the air. Any bacteria that fall onto the plates can then be detected following a suitable period of incubation. These gravitational methods are obviously applicable to many microorganisms, but present problems with viruses. However, more meaningful data can be obtained if force rather than gravity is used to collect airborne particles. A stream of air can be directed onto the surface of a nutrient agar plate (impaction; slit sampler) or bubbled through an appropriate buffer or culture medium (liquid impingement). Various commercial impactor samplers are available. Filtration sampling, where the air is passed through a porous membrane, which is then cultured, can also be used. For experimental evaluation of potential air disinfectants, bacterial or fungal airborne ‘suspensions’ can be created in a closed chamber, and then exposed to the disinfectant, which may be in the form of radiation, chemical vapour or aerosol. The airborne microbial population is then sampled at regular intervals using an appropriate forced-air apparatus such as the slit sampler. With viruses, the air can be bubbled through a suitable liquid medium, which is then subjected to some appropriate virological assay system. In all cases, problems arise in producing a suitable airborne microbial ‘suspension’ and in neutralizing residual disinfectant, which may remain in the air. Solar heating, which can result in a 7-log reduction in viable bacteria over 6 hours, is a possible disinfection consideration for developing countries.
A number of important infectious diseases are spread via microbial contamination of the air. This cross-infection can occur in a variety of situations — hospitals, cinemas, aeroplanes — while main195
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6 Evaluation of preservatives
7 Rapid evaluation procedures
Preservatives are widely employed in the cosmetic and pharmaceutical industries as well as in a variety of other manufacturing industries. While the inhibitory or cidal activity of the chemical to be used as the preservative can be evaluated using an appropriate in vitro test system (see sections 3.2.1 and 8.1.2) its continued activity when combined with the other ingredients in the final manufactured product must be established. Problems clearly exist with some products, where partitioning into various phases may result in the absence of preservative in one of the phases e.g. oil-in-water emulsions where the preservative may partition only into the oily phase, allowing any contaminant microorganisms to flourish in the aqueous phase. In addition, one or more of the components may inactivate the preservative. Some sort of simulated-use challenge test involving the final product is, therefore, required in addition to direct potency testing of the pure preservative. In the challenge test, the final preserved product is deliberately inoculated with a suitable environmental microorganism which may be fungal, e.g. candida, or bacterial, e.g. Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa. For preparations with a high sucrose content, the osmophilic yeast Zygosaccharomyces rouxii is a consideration. The subsequent survival (inhibition), death or growth of the inoculum is then assessed using viable count techniques. Different performance criteria are laid down for injectable and ophthalmic preparations, topical preparations and oral liquid preparations in the British Pharmacopoeia and European Pharmacopoeia, which should be consulted for full details of the experimental procedures to be used. In some instances, the range and/or spectrum of preservation can be extended by using more than one preservative at a time. Thus a combination of parabens (p-hydroxybenzoic acid) with varying water solubilities may protect both the aqueous and oil phases of an emulsion, while a combination of Germall 115 and parabens results in a preservative system with both antibacterial (Germall 115) and antifungal (parabens) activity.
In most of the tests mentioned above, results are not available until visible microbial growth occurs (at least in the controls). This usually takes 24 hours or more. Can the process be accelerated? While the answer is basically yes, only a few ‘rapid’ methods for detecting microbial viability or growth are presently employed in assessing the efficacy of antimicrobials (see Hugo & Russell, 1998). These include epifluorescent and bioluminescence techniques. The former relies on the fact that when exposed to the vital stain acridine orange and viewed under UV light, viable cells fluoresce green or greenish yellow, while dead cells appear orange. With tests involving liquid systems the early growth of viable cells can be assessed by some lightscattering processes while blood culture techniques have classically used the production of CO2 as an indicator of bacterial metabolism and growth. In addition, the availability of molecular techniques, such as quantitative PCR, may be useful in demonstrating the presence or growth of microorganisms that are slow or difficult to culture under usual laboratory conditions, e.g. viruses. This may obviate the need to neutralize residual disinfectant with some assays.
196
8 Evaluation of potential chemotherapeutic antimicrobials Unlike tests for the evaluation of disinfectants where determination of cidal activity is of paramount importance, tests involving potential chemotherapeutic agents (antibiotics) invariably have as their main focus determination of MIC. 8.1 Tests for bacteriostatic activity The historical gradient plates, ditch-plate and cupplate techniques (see Hugo & Russell, 1998) have been replaced by more quantitative techniques such as disc diffusion (Fig. 11.4), broth and agar dilution, and E-tests (Fig. 11.5). All employ chemically defined media (e.g. Mueller-Hinton or IsoSensitest) at a pH of 7.2–7.4, and in the case of solid media, agar plates of defined thickness.
Laboratory evaluation of antimicrobial agents
Fig. 11.4 Disc test with inhibition zones around two (1, 2) of
five discs. While the zone around disc 1 is clear and easy to measure, that around disc 2 is indistinct. While none of the antimicrobials in discs 3, 4 or 5 appear to inhibit the bacterium, synergy (as evidenced by inhibition of growth between the discs) is evident with the antimicrobials in discs 3 and 5. Slight antagonism of the drug in disc 1 by that in disc 3 is evident.
Regularly updated guidelines have been provided by the National Committee for Clinical Laboratory Standards (NCCLS) and are widely used in many countries, although the British Society for Antimicrobial Chemotherapy has produced its own guidelines and testing procedures (see Further reading section). 8.1.1 Disc tests These are really modifications of the earlier cup or ditch-plate procedures where filter-paper discs impregnated with the antimicrobial replace the antimicrobial-filled cups or wells. For disc tests, standard suspensions (e.g. 0.5 McFarland standard) of log phase growth cells are prepared and inoculated onto the surface of appropriate agar plates to form a lawn. Commercially available filter-paper discs containing known concentrations of antimicrobial agent (it is possible to prepare your own discs for use with novel drugs) are then placed on
Fig. 11.5 E-test on an isolate of Candida albicans. Inhibition zone edges are distinct and the MICs for itraconazole (IT) and fluconazole (FL) (0.064 mg/L and 1.5 mg/L, respectively) are easily decipherable.
the dried lawn and the plates are incubated aerobically at 35°C for 18 hours. The density of bacteria inoculated onto the plate should produce just confluent growth after incubation. Any zone of inhibition occurring around the disc is then measured, and after comparison with known standards, the bacterium under test is identified as susceptible or resistant to that particular antibiotic. For novel agents, these sensitivity parameters are only available after extensive clinical investigations are correlated with laboratory-generated data. Disc tests are basically qualitative, although it is possible to get some information on the degree of activity depending on the zone size (Fig. 11.6). Although there are subtle variations of the disc test used in some countries, the basic principles behind the tests remain similar and are based on the original work of Bauer and colleagues. Some techniques employ a control bacterial isolate on each plate so that comparisons between zone sizes around the test and control bacterium can be ascertained (i.e. a disc potency control). Provided that discs are maintained and handled as recommended by the manufacturer, the value of such controls becomes debatable and probably unnecessary. Con197
Chapter 11
≥264.0
32.0
2
C
16.0 3 2 3
3
3
8.0 MIC (mg/L)
B 4.0
3
7 11 12 2
3 2 3
3 2
2
3 2
2 6
3
2.0 A 2 7 10 15 11 5
3 2 2
1.0
0.5
0.25 ≤0.12
Fig. 11.6 A scattergram and regression 2 3 8 16 17 21 13 11 10 2 2 2
3 7 8 7 8 4
11 6 13
5 6 12 6 71616 12 20 9 9 4
2
≤8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 ≥38 Zone diameter (mm)
trol strains of bacteria are available which should have inhibition zones of a given diameter with stipulated antimicrobial discs. Use of such controls endorses the suitability of the methods (e.g. medium, inoculum density, incubation conditions) employed. For slow-growing microorganisms, the incubation period can be extended. Problems arise with disc tests where the inoculum density is inappropriate (e.g. too low resulting in an indistinct edge to the inhibition zone following incubation), or where the edge is obscured by the sporadic growth of cells within the inhibition zone, i.e. the initial inoculum although pure contains cells expressing varying levels of susceptibility — socalled heterogeneity. As the distance from the disc increases, there is a logarithmic reduction in the antimicrobial concentration; the result is that small differences in zone diameter with antimicrobials 198
line analysis correlating zone diameters and MICs. The breakpoints of susceptible (MIC £ 2.0 mg/L, zone diameter ≥ 21 mm) and resistant (MIC ≥ 8.0 mg/L, zone £ 15 mm) are shown by the dotted lines. For a complete correlation between MICs and zone diameter, all susceptible, intermediate and resistant isolates should fall in boxes A, B and C, respectively. Errors (correlations outside these boxes) occur (courtesy of Dr Z. Hashmi).
(e.g. vancomycin) which diffuse poorly through solid media may represent significantly different MICs. Possible synergistic or antagonistic combinations of antimicrobials can often be detected using disc tests (Fig. 11.4). 8.1.2 Dilution tests These usually employ liquid media but can be modified to involve solid media. Doubling dilutions — usually in the range 0.12–256 mg/L — of the antimicrobial under test are prepared in a suitable broth medium, and a volume of log phase cells is added to each dilution to result in a final cell density of around 5 ¥ 105 CFU/ml. After incubation at 35°C for 18 hours, the concentration of antimicrobial contained in the first clear tube is read as the MIC. Needless to say, dilution tests require a number of
Laboratory evaluation of antimicrobial agents
controls, e.g. sterility control, growth control, and the simultaneous testing of a bacterial strain with known MIC to show that the dilution series is correct. Endpoints with dilution tests are usually sharp and easily defined, although ‘skipped’ wells (inhibition in a well with growth either side) and ‘trailing’ (a gradual reduction in growth over a series of wells) may be encountered. The latter is especially evident with antifungal tests (see below). Nowadays, the dilution test for established antimicrobials has been simplified by the commercial availability of 96-well microtitre plates which have appropriate antimicrobial dilutions frozen or lyophilized onto wells in the plate. The appropriate antibacterial suspension (in 200–400-ml volumes) is simply added to each well, the plate is incubated as before, and the MIC is read. Dilution tests can also be carried out using a series of agar plates containing known antimicrobial concentrations. Appropriate bacterial suspensions are inoculated onto each plate and the presence or absence of growth is recorded after suitable incubation. Most clinical laboratories now employ agar dilution breakpoint testing methods. These are essentially truncated agar dilution MIC tests employing only a small range of antimicrobial concentrations around the critical susceptible/resistant cut off levels. Many automated identification and sensitivity testing machines now utilize a liquid (broth) variant of the agar breakpoint procedure. Similar breakpoint antimicrobial concentrations are used with the presence or absence of growth being recorded by some automated procedure (e.g. light-scattering, colour change) after a suitable incubation period.
of lines and figures denoting MIC values (Fig. 11.5). The nylon strips are placed antimicrobial side down on the freshly prepared bacterial lawn and, after incubation, the MIC is determined by noting where the ellipsoid (pear-shaped) inhibition zone crosses the strip (Fig. 11.5). For most microorganisms, there appears to be excellent correlation between dilution and E-test MIC results.
8.1.3 E-tests
8.2 Tests for bactericidal activity
Perhaps the most convenient and presently accepted method of determining bacterial MICs, however, is the E (Epsilometer)-test. Basically this is performed in a similar manner to the disc test except that nylon strips that have a linear gradient of antimicrobial lyophilized on one side are used instead of the filter-paper impregnated antimicrobial discs. On the other side of the nylon strip are a series
Minimum bactericidal concentration (MBC) testing is required for the evaluation of novel antimicrobials. The MBC is the lowest concentration (in mg/L) of antimicrobial that results in ≥99.9% killing of the bacterium under test. The 99.9% cutoff is an arbitrary in vitro value with 95% confidence limits that has uncertain clinical relevance. MBCs are determined by spreading 0.1-ml
8.1.4 Problematic bacteria With some of the emerging antimicrobial-resistant bacterial pathogens, e.g. vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate S. aureus (VISA), the standard methodology described above may fail to detect the resistant phenotype. This is due to a variety of factors including heterogeneous expression of resistance (e.g. MRSA, VISA), poor agar diffusion of the antimicrobial (e.g. vancomycin) and slow growth of resistant cells (e.g. VISA). Disc tests are unsuitable for VRE that should have MICs determined by Etest or dilution techniques. With MRSA, a heavier inoculum should be used in tests and 2–4% additional salt (NaCl) included in the medium with incubation for a full 48 hours. Reducing the incubation temperature to 30°C may also facilitate detection of the true MIC value. Although 100% of MRSA cells may contain resistance genes, the phenotype may only be evident in a small percentage of cells under the usual conditions employed in sensitivity tests. Expression is enhanced at lower temperatures and at higher salt concentrations. With VISA, MIC determinations require incubation for a full 24 hours or more because of the slower growth rate of resistant cells.
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(100-ml) volumes of all clear (no growth) tubes from a dilution MIC test onto separate agar plates (residual antimicrobial in the 0.1-ml sample is ‘diluted’ out over the plate). After incubation at 35°C overnight (or longer for slow-growing bacteria), the numbers of colonies growing on each plate are recorded. The first concentration of drug that produces 100-fold increase in killing of the combination compared with either drug alone. Antagonism is defined as at least a 100-fold decrease in killing of the combination when compared with the most active agent alone, while an additive or autonomous combined effect results in a 32 mg/ml. The mechanism of glycopeptide resistance is poorly understood, but strains show longer doubling times and decreased susceptibility to lysostaphin. Increased quantities of PBP2 and PBP2¢ and cell wall precursors are presumed to trap vancomycin, while amidation of glutamine residues in cell wall muropeptides reduces the cross-linking and consequently the number of vancomycin target molecules.
5.1 MRSA and reduced glycopeptide susceptibility
6 Resistance to aminoglycoside antibiotics
There is major concern that high-level, VanA-type resistance could transfer to staphylococci, particularly MRSA. Experimental transfer of the enterococcal VanA system to S. aureus on the skin of mice has been reported, but other mechanisms resulting in intermediate-level resistance occur in clinical isolates. In the 1960s and 1970s MRSA was not feared because several other treatment options existed, including use of tetracyclines, macrolides and aminoglycosides. But multiple resistance was accumulating and by the 1980s empiric therapy of staphylococcal infections, particularly nosocomial sepsis, was changed to the glycopeptide antibiotic
The aminoglycosides are hydrophilic sugars possessing a number of amino and hydroxy substituents. The amine groups are protonated at biological pH and it is the polycationic nature of the molecules that affords them their affinity for nucleic acids, particularly the acceptor (A) site of 16S ribosomal RNA. Aminoglycoside binding to the A site interferes with the accurate recognition of cognate tRNA by rRNA during translation and may also perturb translocation of the tRNA from the A site to the peptidyl-tRNA site (P site). While high-level resistance in aminoglycoside-producing microorganisms is by methylation of the rRNA, this
224
Bacterial resistance to antibiotics
Vancomycin
O
N
N
N
H
H H
O
H N
R
H
O
H N
H
N
O H
O
Stable complex between vancomycin and D-Ala-D-Ala
Vancomycin
O N
N
N
H
H H
Fig. 13.4 Mechanism of high-level
vancomycin resistance. Hydrogen bonds are denoted by dashed lines. The key hydrogen bond present in the stable complex with D-Ala-D-Ala, but missing in the unstable complex with D-Ala-D-Lac, is shown in heavy type.
O R
O
H N H
O O
H
N
O H
Unstable complex between vancomycin and D-Ala-D-Lac
is NOT the mechanism of resistance in previously susceptible strains. The most common mechanism for clinical aminoglycoside resistance is their structural modification by enzymes expressed in resistant organisms, which compromises their ability to
interact with rRNA. There are three classes of these enzymes: aminoglycoside phosphatases (APHs), aminoglycoside nucleotidyltransferases (ANTs) and aminoglycoside acetyltransferases (AACs). Within each class, there are enzymes with differing 225
Chapter 13 AAC(6’) AAC(3)
ANT(4’)
AAC(1)
NH2 O 1¢
HO HO 3¢
APH(3’)
APH(2’’)
H2N 3
NH2 O
HO
1 NH2 O HO
AAC(2’)
1¢¢ O
ANT(2’’) 3¢¢
NH2 OH OH
Fig. 13.5 Structure of kanamycin B showing system of ring
numbering and sites of action of some aminoglycosidemodifying enzymes. AAC, aminoglycoside acetyltransferase; ANT, aminoglycoside nucleotidyltransferase and APH, aminoglycoside phosphotransferase.
specificities around the sugars. There are four ANTs [ANT(6), ANT(4¢), ANT(3≤) and ANT(2≤)], seven APHs [APH(3¢), APH(2≤), APH(3≤), APH(6), APH(9), APH(4) and APH(7≤)] and four AACs [AAC(2¢), AAC(6¢), AAC(1) and AAC(3)]. There is also a bifunctional enzyme, AAC(6¢)-AAC(2≤). Aminoglycosides are typically susceptible to attack by multiple enzymes (Fig. 13.5). Attempts to circumvent these modifying enzymes have centred on structural modifications. Examples include tobramycin which lacks the 3¢-hydroxyl group and is thus not a substrate for APH(3¢) and amikacin which has an acylated N-1 group and is not a substrate for several modifying enzymes. Other strategies are exemplified by experimental compounds such as 3¢-oxo-kanamycin. This molecule is a substrate for APH(3¢), but the phosphorylation product is unstable and regenerates the original antibiotic.
7 Resistance to tetracycline antibiotics Chlortetracycline and oxytetracycline were discovered in the late 1940s and studies of representative populations before their widespread use suggests that emergence of resistance is a relatively modern event. More than 60% of Shigella flexneri isolates are resistant to tetracycline; resistant isolates of Salmonella enterica serovar typhimurium are becoming more common and among Gram-positive 226
species, approximately 90% of MRSA strains and 60% of multiply resistant Streptococcus pneumoniae are now tetracycline-resistant. The major mechanisms of resistance are efflux and ribosomal protection. One exception is the tet(X) gene that encodes an enzyme which modifies and inactivates the tetracycline molecule, although this does not appear to be clinically significant. The Tet efflux proteins belong to the major facilitator superfamily (MFS). These proteins exchange a proton for a tetracycline–cation (usually Mg2+) complex, reducing the intracellular drug concentration and protecting the target ribosomes in the cell. In Gramnegative bacteria, the efflux determinants comprise divergently oriented efflux and repressor proteins that share overlapping promoter and operator regions. In the absence of a tetracycline–Mg2+ complex, the repressor protein binds and blocks transcription of both genes. Drug binding alters the conformation of the repressor so that it can no longer bind the DNA operator region and block transcription. This method of regulation probably applies to all of the Gram-negative efflux systems including tet(A), tet(C), tet(D), tet(E), tet(G) and tet(H). No repressor proteins have been identified in the Gram-positive tet(K) or tet(L) genes and regulation of plasmid-borne tetracycline resistance appears to be by translational attenuation, involving stemloop mRNA structures and tetracycline-induced unmasking of the ribosome binding site permitting translation of the efflux protein. Regulation of chromosomal tet(L) expression involves tetracycline-promoted stalling of the ribosomes during translation of early codons of the leader peptide, which allows re-initiation of translation at the ribosome binding site for the structural gene. Ribosomal protection is mediated by cytoplasmic proteins that inhibit tetracycline and also confer resistance to doxycycline and minocycline. These proteins share homology with the elongation factors EF-Tu and EF-G, and expression of Tet(M) and Tet(O) proteins appears to be regulated. A 400-bp region upstream from the coding region for tet(O) is needed for full expression, but the mechanism(s) has not been characterized. The widespread emergence of efflux- and ribosome protection-based resistance to first- and second-generation
Bacterial resistance to antibiotics N(CH3)2
N(CH3)2 OH
OH
O
OH OH O
CONH2
Minocycline N(CH3)2
N(CH3)2 OH
O (CH3)2N
N H
OH
O
OH OH O
CONH2
9-(N,N-Dimethylglycylamido)-minocycline N(CH3)2
H3C H3C
H N CH3
N(CH3)2 OH
O N H
OH
O
OH OH O
CONH2
9-(t-butylglycylamido)-minocycline (Tigicycline GAR-936) Fig. 13.6 Structural modifications at the 9 position of the
tetracycline antibiotic minocycline conferring increased stability against resistance mechanisms.
tetracyclines has prompted the development of the 9-glycinyltetracyclines (9-glycylcyclines). 9-Aminoacylamido derivatives of minocycline have similar activity to earlier compounds; however, when the acyl group is modified to include an N,Ndialkylamine or 9-t-butyl-glycylamido moiety (Fig. 13.6), antimicrobial activity is retained and the compounds are active against strains containing tet genes responsible for resistance by efflux and ribosomal protection.
8 Resistance to fluoroquinolone antibiotics Fluoroquinolones bind and inhibit two bacterial topoisomerase enzymes: DNA gyrase (topoisomerase II) which is required for DNA supercoiling, and topoisomerase IV which is required for strand separation during cell division. DNA gyrase tends to be the major target in Gram-negative bacteria,
whereas both topoisomerases are inhibited in Gram-positive bacteria. Each topoisomerase is termed a heterotetramer, being composed of two copies of two different subunits designated A and B. The A and B subunits of DNA gyrases are encoded by gyrA and gyrB, respectively, whilst topoisomerase IV is encoded by parC and parE (grlA and grlB in S. aureus). Mutations in gyrA, particularly involving substitution of a hydroxyl group with a bulky hydrophobic group, induce conformational changes such that the fluoroquinolone can no longer bind. Mutations have also been detected in the B subunit, but these are probably less important. Alterations involving Ser80 and Glu84 of S. aureus grlA and Ser79 and Asp83 of S. pneumoniae parC have led to quinolone resistance. Like GyrB, mutations in ParE leading to resistance are not common. While changes in GyrA and ParC give resistance to the older fluoroquinolones, MIC values do not always rise above clinically defined breakpoints for newer agents such as gemifloxacin and moxifloxacin. Topoisomerases are located in the cytoplasm and thus fluoroquinolones must cross the cell envelope to reach their target. Changes in outer-membrane permeability have been associated with resistance in Gram-negative bacteria, but permeability does not appear to be an issue with Gram-positive species. Efflux, however, does make a contribution to resistance, mainly low level, in both Grampositive and Gram-negative bacteria. The NorAmediated efflux system in S. aureus was characterized in 1990. It is expressed weakly in wild-type strains and resistance is thought to occur via mutations leading to increased expression of norA. NorA is a member of the major facilitator superfamily and homologues are also present in Streptococcus pneumoniae and Bacillus sp. There is a tendency for it to be more effective for hydrophilic fluoroquinolones, but there is no strict correlation. Fluoroquinolones are now being used for treating Mycobacterium avium and multidrug-resistant M. tuberculosis and efflux-mediated resistance has been identified. A number of efflux pumps have been identified among Gram-negative bacteria, including AcrA in E. coli, which is regulated in part by the multiple antibiotic resistance (Mar) operon (section 16.3). 227
Chapter 13
9 Resistance to macrolide, lincosamide and streptogramin (MLS) antibiotics N
N
Although chemically distinct, members of the MLS group of antibiotics all inhibit bacterial protein synthesis by binding to a target site on the ribosome. Gram-negative bacteria are intrinsically resistant due to the permeability barrier of the outer membrane, and three resistance mechanisms have been described in Gram-positive bacteria. Target modification, involving adenine methylation of domain V of the 23S ribosomal RNA, is the most common mechanism. The adenine-N6-methyltransferase, encoded by the erm gene, results in resistance to erythromycin and other macrolides (including the azalides), as well as the lincosamides and group B streptogramins. Streptogramin A-type antibiotics are unaffected and streptogramin A/B combinations remain effective. Expression of the erm gene may be constitutive or inducible. When expression is inducible, resistance is seen only against 14- and 15-membered macrolides; lincosamide and streptogramin antibiotics remain active. Telithromycin (Fig. 13.7), the first of a new class of ketolide agents in the MLS family, does not induce MLS resistance and also retains activity against domain V-modified ribosomes and inhibition of protein synthesis through strong interaction with domain II. The second resistance mechanism is efflux. Expression of the mef gene confers resistance to macrolides only, whereas msr expression results in resistance to macrolides and streptogramins. Efflux-mediated resistance of S. aureus to streptogramin A antibiotics is also conferred by vga and vgaB gene products. A third resistance mechanism, involving ribosomal mutation, has been reported in a small number of clinical isolates of S. pneumoniae.
10 Resistance to chloramphenicol Chloramphenicol inhibits protein synthesis by binding the 50S ribosomal subunit and preventing the peptidyltransferase step. Decreased outermembrane permeability and active efflux have been identified in Gram-negative bacteria; however, the major resistance mechanism is drug inactivation by chloramphenicol acetyltransferase. This occurs in 228
N
O
CH3
O N O O
O O
O O
N Fig. 13.7 Structure of telithromycin, a ketolide macrolide
antibiotic that retains activity against 23S domain V modified ribosomal RNA.
both Gram-positive and Gram-negative species, but the cat genes, typically found on plasmids, share little homology.
11 Resistance to the oxazolidinone antibiotics Linezolid is the first of a new class of oxazolidinone antimicrobials with a novel target in protein synthesis. Linezolid does not interfere with translation initiation at the stage of mRNA binding or formation of 30S pre-initiation complexes, rather it involves binding the 50S rRNA. Its affinity for 50S rRNA from Gram-positive bacteria is twice that for the corresponding molecule in Gram-negative bacteria and as such linezolid has been approved for treating various Gram-positive infections, including MRSA. Resistance is appearing, although rare at present. Mutation in the central loop of domain V of the component 23S rRNA subunit appears to be
Bacterial resistance to antibiotics
the main mechanism, including a G2576T mutation in three isolates of linezolid-resistant MRSA.
12 Resistance to trimethoprim Trimethoprim competitively inhibits dihydrofolate reductase (DHFR) and resistance can be caused by overproduction of host DHFR, mutation in the structural gene for DHFR and acquisition of the dfr gene encoding a resistant form. There are at least 15 DHFR enzyme types based on sequence homology and acquisition of dfr genes encoding alternative DHFR of type I, II or V is the most common mechanism of trimethoprim resistance among the Enterobacteriaceae.
13 Resistance to mupirocin Nasal carriage of MRSA strains has been identified as an important target for infection control protocols aimed at reducing spread and acquisition. Mupirocin (pseudomonic acid A) is an effective topical antimicrobial used in MRSA eradication. It is an analogue of isoleucine that competitively binds isoleucyl-tRNA synthetase (IRS) and inhibits protein synthesis. Low-level resistance (MIC 4– 256 mg/ml) is usually due to mutation of the host IRS, whereas high-level resistance (MIC >512 mg/ml) is due to acquisition of a distinct IRS that is less sensitive to inhibition. The mupA gene, typically carried on transferable plasmids, is found in S. aureus and coagulase-negative staphylococci, and encodes an IRS with only 30% homology to the mupirocin-sensitive form.
14 Resistance to peptide antibiotics — polymyxin Many peptide antibiotics have been described and can be broadly classified as non-ribosomally synthesized peptides; they include the polymyxins, bacitracins and gramicidins as well as the glycopeptides (section 5) and the ribosomally synthesized peptides such as the antimicrobial peptides of the innate immune system. Polymyxins and other
cationic antimicrobial peptides have a selfpromoted uptake across the cell envelope and perturb the cytoplasmic membrane barrier. Addition of a 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety to the phosphate groups on the lipid A component of Gram-negative lipopolysaccharide has been implicated in resistance to polymyxin. Details of the pathway for L-Ara4N biosynthesis from UDP glucuronic acid, encoded by the pmr operon, are emerging.
15 Resistance to anti-mycobacterial therapy The nature of mycobacterial infections, particularly tuberculosis, means that chemotherapy differs from other infections. Organisms tend to grow slowly (long generation time) in a near dormant state with little metabolic activity. Hence, a number of the conventional antimicrobial targets are not suitable. Isoniazid is bactericidal, reducing the count of aerobically growing organisms. Pyrazinamide is active only at low pH, making it well suited to killing organisms within necrotic foci early in infection, but less useful later on when these foci have reduced in number. Rifampicin targets slowgrowing organisms. Resistance mechanisms have now been described and multiple resistance poses a serious threat to health. Current treatment regimens do result in a high cure rate and the combination of agents makes it highly unlikely that there will be a spontaneous resistant isolate to all the components. Problems most commonly occur in patients who receive inadequate therapy which provides a serious selection advantage. Resistance can occur to single agents and subsequently to multiple agents. Resistance to rifampicin arises from mutation in the beta subunit of RNA polymerase encoded by rpoB and resistant isolates show decreased growth rates. Modification of the catalase gene katG results in resistance to isoniazid, mainly by reduced or absent catalase activity. Catalase activity is absolutely required to convert isoniazid to the active hydrazine derivative. Interestingly, animal model studies suggest that M. tuberculosis strains in which the katG gene is inactivated are attenuated compared with wild-type 229
Chapter 13
strains. Low-level rifampicin resistance can be obtained by point mutations in inhA leading to its overexpression. Pyrazinamide is a pro-drug requiring pyrazinamidase to produce the active pyrazinoic acid. Most cases of resistance are due to mutations in the pyrazinamidase gene (pncA), but gene inactivation by the insertion sequence IS6110 has been reported. Streptomycin resistance can arise through mutations in rrs and rpsL which affect streptomycin binding. However, these account for only half of the resistant isolates, so further resistance mechanisms await definition. Ethambutol resistance has been noted in M. tuberculosis and other species such as M. smegmatis. Ethambutol inhibits the polymerization of arabinan in the arabinogalactan and lipoarabinomannan of the mycobacterial cell wall and one of its likely targets is the family of arabinosyltransferases encoded by the emb locus. Missense mutations in the embB gene in this locus confer resistance to ethambutol.
16 Multiple drug resistance 16.1 R-factors Several issues of multiple drug resistance have already been raised in this chapter. Notable examples are MRSA, which can harbour both small cryptic plasmids and larger plasmids encoding resistance to antiseptics, disinfectants, trimethoprim, penicillin, gentamicin, tobramycin and kanamycin, and multidrug-resistant M. tuberculosis. Of equal concern are instances where isolates can become resistant to multiple, chemically distinct agents in a single biological event. One of the earliest examples was in Japan in 1959. Previously sensitive E. coli became resistant to multiple antibiotics through acquisition of a conjugative plasmid (R-factor) from resistant Salmonella and Shigella isolates. A number of R-factors have now been characterized including RP4, encoding resistance to ampicillin, kanamycin, tetracycline and neomycin, found in P. aeruginosa and other Gram-negative bacteria; R1, encoding resistance to ampicillin, kanamycin, sulphonamides, chloramphenicol and streptomycin, found in Gram-negative bacteria and pSH6, encoding resistance to gentamicin, trimethoprim and kanamycin, found in S. aureus. 230
16.2 Mobile gene cassettes and integrons Many Gram-negative resistance genes are located in gene cassettes. One or more of these cassettes can be integrated into a specific position on the chromosome termed an integron. More than 60 cassettes have been identified, each comprising only a promotor-less single gene (usually antibiotic resistance) and a 59-base element forming a specific recombination site. This recombination site confers mobility because it is recognized by specific recombinases encoded by integrons that catalyse integration of the cassette into a specific site within the integron. Thus, integrons are genetic elements that recognize and capture multiple mobile gene cassettes. As the gene typically lacks a promoter, expression is dependent on correct orientation into the integron to supply the upstream promoter. Four classes of integron have been identified, although only one member of class 3 has been described and class 4 integrons are limited to Vibrio cholerae. Analysis of the resistant Shigella strains isolated in Japan has shown that some of the conjugative plasmids included an integron with one or two integrated cassettes. 16.3 Chromosomal multiple-antibiotic resistance (Mar) locus The multiple-antibiotic resistance (mar) locus was first described in Escherichia coli by Stuart Levy and colleagues at Tufts University and has since been recognized in other enteric bacteria. The locus consists of two divergently transcribed units, marC and marRAB. Little is known of marC and marB; however, marR encodes a repressor of the operon, and marA encodes a transcriptional activator affecting expression of more than 60 genes. Increased expression of the MarRAB operon resulting from mutations in marO or marR, or from inactivation of MarR following exposure to inducing agents such as salicylate, leads to the Mar phenotype. This phenotype is characterized by resistance to structurally unrelated antibiotics, organic solvents, oxidative stress and chemical disinfectants. A number of effector mechanisms have been identified, including increased expression of the acrAB-tolC multidrug efflux system (section 16.4) and the soxRS regulon.
Bacterial resistance to antibiotics
16.4 Multidrug efflux pumps Whereas some efflux pumps excrete only one drug or class of drugs, a multidrug efflux pump can excrete a wide range of compounds where there is often little or no chemical similarity between the substrates. One common characteristic may be agents with a significant hydrophobic domain. For this reason, hydrophilic compounds such as the aminoglycoside antibiotics are not exported by these systems. A distinction needs to be drawn between those efflux systems, typically in Grampositive bacteria, that pump their substrate across the cytoplasmic membrane, such as the QacA and Smr pumps which both export quaternary ammonium compounds and basic dyes, and those which efflux across the cytoplasmic and outer membranes of Gram-negative bacteria. There are some examples of single membrane systems in Gram-negative bacteria, such as the EmrE protein in E. coli, but they are not of great clinical significance. The majority of Gram-negative pumps span both membranes and include the AcrAB-TolC system in E. coli and the MexAB-OprM system in P. aeruginosa. Genomic analyses are revealing numerous homologues. Using the MexAB-OprM system as the prototypic example (Fig. 13.8), MexA is the linker protein and MexB is in the cytoplasmic membrane. MexB is a resistance-nodulation-division (RND) family member and is predicted to be a proton antiporter with 12 membrane-spanning a-helices. OprM shows homology with outer-membrane channels of systems thought to export such diverse molecules as nodulation signals and alkaline proteases. Mutations in regulatory genes such as nalB cause overexpression of MexAB-OprM and consequently multidrug resistance. MexB is a proton antiporter and efflux by this and other members of the RND family is energized by proton motive force. This contrasts with mammalian multidrug efflux pumps (MDR) that are powered by ATP hydrolysis.
17 Clinical resistance — MIC values, breakpoints, phenotype and outcome The resistance mechanisms described in this chapter typically lead to an increase in MIC value,
Antibiotic
OM
OprM channel
MexA fusion protein
H+
MexB efflux transporter
Periplasm
CM
H+ Fig. 13.8 Schematic diagram of the MexAB-OprM efflux
pump from Pseudomonas aeruginosa. (Kindly supplied by K. Poole.)
although it should be remembered that this does not always equate with clinical failure. If the MIC value remains below the breakpoint value, which can itself be difficult to determine, then the antibiotic will remain effective in the clinic. But such arguments assume that MIC values, which are typically determined when the isolate is growing in complex, sensitivity-test broth, equate with the sensitivity of the organism when growing in the many subtly different environments encountered during infection in vivo. Unfortunately, the antibiotic literature contains numerous examples of treatment failures despite apparent sensitivity in the test tube and this resistance is referred to as being phenotypic. Put in other words, resistance is a consequence of the adaptation of the organism to grow and survive within the in vivo environment and subculture into conventional laboratory growth medium rarely shows the existence of resistant mutants. There are several key factors at play here, particularly slow/no growth, nutrient depletion and mode of growth. There are numerous papers showing a tendency for nutrient depletion, that is restricted or non-availability of an important nutrient, and slow/no growth to be associated with reduced susceptibility to antibiotics and biocides. There is now increasing concern over the role played by microbial biofilms in infection. These include the well-known examples of medical device231
Chapter 13
related infections, such as those associated with artificial joints, prosthetic heart valves and catheters. Many chronic infections, not related to medical devices, are also due to bacteria either not growing and relatively dormant or growing slowly as biomasses or adherent biofilms on mucosal surfaces. A bacterial biofilm is typically defined as a population of cells growing as a consortium on a surface and enclosed in a complex exopolymer matrix. Commonly in the wider environment but less so in infections, the population is mixed and also of heterogeneous physiologies. Growth as a biofilm almost always leads to a large increase in resistance to antimicrobial agents, including antibiotics, biocides and preservatives, compared with cultures grown in suspension (planktonic) in conventional liquid media, but there is no generally agreed mechanism to account for this resistance. Although there is general acceptance that there are numerous planktonic phenotypes, many papers refer to ‘the biofilm phenotype’, implicitly assuming (wrongly) that there is only one. Those same parameters known to influence planktonic physiology and antibiotic susceptibility, including growth rate and/or specific nutrient limitation, also apply to biofilm physiology and antibiotic susceptibility. The general resistance of biofilms is clearly phenotypic. The well characterized resistance mechanisms described above — lack of antibiotic penetration, inactivation, efflux and repair — make contributions in some circumstances. However, compelling evidence that they are uniquely responsible for biofilm resistance is lacking. Reduced growth rate probably has an involvement, particularly in that it is associated with responses to stress. During stress responses, key structures are protected and cellular processes close down to a state of dormancy, and it has been proposed that exceptional vegetative cell dormancy is the basic explanation of biofilm resistance.
18 Concluding comments It has been said that in the early years of the 21st
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century we are in an interim between the first antibiotic era, exemplified by the b-lactams, macrolides, tetracyclines, aminoglycosides and fluoroquinolones, and a second era of new agents directed against targets waiting to be revealed by genomics and proteomics research. In this interim, the goals must be to reduce antibiotic usage and encourage the return of a susceptible human commensal flora. Finally, it must be remembered that antibiotics are not man’s invention. Microorganisms have used them in attack and counter-attack against each other for billions of years. Our efforts over the last 60 years seem trivial in comparison.
19 Further reading Alekshun, M. N. & Levy, S. B. (1999) The mar regulon: multiple resistance to antibiotics and other toxic chemicals. Trends Microbiol, 7, 410–413. Chopra, I. & Roberts, M. (2001) Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev, 65, 232–260. Fluit, A. C., Visser, M. R. & Schmitz, F. J. (2001) Molecular detection of antimicrobial resistance. Clin Microbiol Rev, 14, 836–871. Gillespie, S. H. (2002) Evolution of drug resistance in Mycobacterium tuberculosis: clinical and molecular perspective. Antimicrob Agents Chemother, 46, 267–274. Kotra, L. P., Haddad, J. & Mobashery, S. (2000) Aminoglycosides: perspectives on mechanism of action and resistance and strategies to counter resistance. Antimicrob Agents Chemother, 44, 3249–3256. Martinez, J. L. & Baquero, F. (2000) Mutation frequencies and antibiotic resistance. Antimicrob Agents Chemother, 44, 1771–1777. Murray, B. E. (2000) Vancomycin-resistant enterococcal infections. N Engl J Med, 342, 710–721. Nikaido, H. (1996) Multidrug efflux pumps of gramnegative bacteria. J Bacteriol 178, 5853–5859. Philippon, A., Arlet, G. & Jacoby, G. A. (2002) Plasmiddetermined AmpC-type b-lactamases. Antimicrob Agents Chemother, 46, 1–11. Recchia, G. D. & Hall, R. M. (1997) Origins of the mobile gene cassettes found in integrons. Trends Microbiol, 5, 389–394.
Chapter 14
Clinical uses of antimicrobial drugs Roger Finch 1 Introduction 2 Principles of use of antimicrobial drugs 2.1 Susceptibility of infecting organisms 2.2 Host factors 2.3 Pharmacological factors 2.4 Drug resistance 2.4.1 Multidrug resistance 2.5 Drug combinations 2.6 Adverse reactions 2.7 Superinfection 2.8 Chemoprophylaxis 3 Clinical use 3.1 Respiratory tract infections 3.1.1 Upper respiratory tract infections 3.1.2 Lower respiratory tract infections
3.2 Urinary tract infections 3.2.1 Pathogenesis 3.2.2 Drug therapy 3.3 Gastrointestinal infections 3.4 Skin and soft tissue infections 3.5 Central nervous system infections 3.6 Fungal infections 3.7 Medical device-associated infections 4 Antibiotic policies 4.1 Rationale 4.2 Types of antibiotic policies 4.2.1 Free prescribing policy 4.2.2 Restricted reporting 4.2.3 Restricted dispensing 5 Further reading
1 Introduction
proved useful in the prevention of infection in various high-risk circumstances; this applies especially to patients undergoing various surgical procedures where peri-operative antibiotics have significantly reduced postoperative infectious complications. The advantages of effective antimicrobial chemotherapy are self-evident, but this has led to a significant problem in ensuring that they are always appropriately used. Surveys of antibiotic use have demonstrated that more than 50% of antibiotic prescribing can be inappropriate; this may reflect prescribing in situations where antibiotics are either ineffective, such as viral infections, or that the selected agent, its dose, route of administration or duration of use are inappropriate. Of particular concern is the unnecessarily prolonged use of antibiotics for surgical prophylaxis. Apart from being wasteful of health resources, prolonged use encourages superinfection by drug-resistant organisms and unnecessarily increases the risk of adverse drug reactions. Thus, it is essential that the clinical use of these agents be based on a clear understanding of the principles that have evolved to ensure safe, yet effective, prescribing.
The worldwide use of antimicrobial drugs continues to rise; in 2000 these agents accounted for an expenditure of approximately £25 billion. In the UK prescribing in general practice accounts for approximately 90% of all antibiotics and largely involves oral and topical agents. Hospital use accounts for the remaining 10% of antibiotic prescribing with a much heavier use of injectable agents. Although this chapter is concerned with the clinical use of antimicrobial drugs, it should be remembered that these agents are also extensively used in veterinary practice and, to a diminishing extent, in animal husbandry as growth promoters. In humans the therapeutic use of anti-infectives has revolutionized the management of most bacterial infections, many parasitic and fungal diseases and, with the availability of aciclovir and a growing number of anti-retroviral agents (see Chapters 5 and 10), selected herpesvirus infections and human immunodeficiency virus (HIV) infection, respectively. Although originally used for the treatment of established bacterial infections, antibiotics have
233
Chapter 14
Further information about the properties of antimicrobial agents described in this chapter can be found in Chapter 10.
2 Principles of use of antimicrobial drugs 2.1 Susceptibility of infecting organisms Drug selection should be based on knowledge of its activity against infecting microorganisms. Selected organisms may be predictably susceptible to a particular agent, and laboratory testing is therefore rarely performed. For example, Streptococcus pyogenes is uniformly sensitive to penicillin. In contrast, the susceptibility of many Gram-negative enteric bacteria is less predictable and laboratory guidance is essential for safe prescribing. The susceptibility of common bacterial pathogens and widely prescribed antibiotics is summarized in Table 14.1. It can be seen that, although certain bacteria are susceptible in vitro to a particular agent, use of that drug may be inappropriate, either on pharmacological grounds or because other less toxic agents are preferred. 2.2 Host factors In vitro susceptibility testing does not always predict clinical outcome. Host factors play an important part in determining outcome and this applies particularly to circulating and tissue phagocytic activity. Infections can progress rapidly in patients suffering from either an absolute or functional deficiency of phagocytic cells. This applies particularly to those suffering from various haematological malignancies, such as the acute leukaemias, where phagocyte function is impaired both by the disease and also by the use of potent cytotoxic drugs which destroy healthy, as well as malignant, white cells. Under these circumstances it is essential to select agents that are bactericidal, as bacteriostatic drugs, such as the tetracyclines or sulphonamides, rely on host phagocytic activity to clear bacteria. Widely used bactericidal agents include the aminoglycosides, broad-spectrum penicillins, the cephalosporins and quinolones (see Chapter 10). In some infections the pathogenic organisms are 234
located intracellularly within phagocytic cells and, therefore, remain relatively protected from drugs that penetrate cells poorly, such as the penicillins and cephalosporins. In contrast, erythromycin, rifampicin and the fluoroquinolones readily penetrate phagocytic cells. Legionnaires’ disease is an example of an intracellular infection and is treated with erythromycin with or without rifampicin. 2.3 Pharmacological factors Clinical efficacy is also dependent on achieving satisfactory drug concentrations at the site of the infection; this is influenced by the standard pharmacological factors of absorption, distribution, metabolism and excretion. If an oral agent is selected, gastrointestinal absorption should be satisfactory. However, it may be impaired by factors such as the presence of food, drug interactions (including chelation), or impaired gastrointestinal function either as a result of surgical resection or malabsorptive states. Although effective, oral absorption may be inappropriate in patients who are vomiting or have undergone recent surgery; under these circumstances a parenteral agent will be required and has the advantage of providing rapidly effective drug concentrations. Antibiotic selection also varies according to the anatomical site of infection. Lipid solubility is of importance in relation to drug distribution. For example, the aminoglycosides are poorly lipidsoluble and although achieving therapeutic concentrations within the extracellular fluid compartment, penetrate the cerebrospinal fluid (CSF) poorly. Likewise the presence of inflammation may affect drug penetration into the tissues. In the presence of meningeal inflammation, b-lactam agents achieve satisfactory concentrations within the CSF, but as the inflammatory response subsides drug concentrations fall. Hence it is essential to maintain sufficient dosaging throughout the treatment of bacterial meningitis. Other agents such as chloramphenicol are little affected by the presence or absence of meningeal inflammation. Therapeutic drug concentrations within the bile duct and gall bladder are dependent upon biliary excretion. In the presence of biliary disease, such as gallstones or chronic inflammation, the drug
+, Sensitive; R, resistant; ±, some strains resistant; (
Proteus spp. (indole-positive)
Serratia spp.
Salmonella spp.
Shigella spp.
Pseudomonas spp.
Bacteroides fragilis
Other Bacteroides spp.
Chlamydia spp.
Mycoplasma pneumoniae
Rickettsia spp.
± +
Proteus spp. (indole-negative)
± +
Klebsiella spp.
+ +
Escherichia coli
+ +
Haemophilus influenzae
+ R + R R R R R R R R + + ± R
Neisseria meningitidis
Enterococcus
+* + +* (+) + + + + + + +* ± + + R
Neisseria gonorrhoeae
Streptococcus pyogenes and Streptococcus pneumoniae
R +* R R +* + + + + + +* +* + ± +
± +* + + R R
+* (±) +* (+) (+) + (+) + + + R + + + R
+ (±) + (+) (+) + (+) + + (+) R (+) + + R
± R ± (+) ± + + + + ± R + +* + +
R R ± ± + + + + + R R + + + +
R R R ± ± + + + + R R ± ± + +*
R R + + + + + + + R R ± + + +
R R R ± R + + + + R R R ± + +
R R R ± R R ± + + R R R + + +*
R R ± (+) (+) (+) (+) (+) (+) R R (+) +* + (+)
R R ± (+) (+) (+) (+) (+) (+) R R (±) + + (+)
R R R +* R R R ± + R R R R (+) +*
R R R ± R R + R/± R ± + ± + R R
+ (±) + ± ± R + R/+ ± ± + ± + R R
R R R R R R R R R + R + + R R
R R R R R R R R R + R + + R R
R R R R R R R R R R R + + R R
(±) R
± +
± +
± +
± +
± +
± +
± +
R R
± +
± +
R R
R R
R R
+ +
R R
R R
Cl. perfringens
Staphylococcus aureus (pen. resistant)
+ + + (+) + + + + + + +* +* + ± +
+ (+) + + (±) + + +
), not appropriate therapy; *, rare strains resistant.
235
Clinical uses of antimicrobial drugs
Penicillin V/G Methicillin, flucloxacillin Ampicillin, amoxicillin Ticarcillin Cefazolin Cefamandole, cefuroxime Cefoxitin Cefotaxime, ceftriaxone Ceftazidime Erythromycin Clindamycin Tetracyclines Chloramphenicol Ciprofloxacin Gentamicin, tobramycin, amikacin, netilmicin Sulphonamides Trimethoprim– sulphamethoxazole
Staphylococcus aureus (pen. sensitive)
Table 14.1 Sensitivity of selected bacteria to common antibacterial agents
Chapter 14
concentration may fail to reach therapeutic levels. In contrast, drugs that are excreted primarily via the liver or kidneys may require reduced dosaging in the presence of impaired renal or hepatic function. The malfunction of excretory organs may not only risk toxicity from drug accumulation, but will also reduce urinary concentration of drugs excreted primarily by glomerular filtration. This applies to the aminoglycosides and the urinary antiseptics nalidixic acid and nitrofurantoin, where therapeutic failure of urinary tract infections may complicate severe renal failure. 2.4 Drug resistance Drug resistance may be a natural or an acquired characteristic of a microorganism. This may result from impaired cell wall or cell envelope penetration, enzymatic inactivation, altered binding sites or active extrusion from the cell as a result of efflux mechanisms (Chapter 13). Acquired drug resistance may result from mutation, adaptation or gene transfer. Spontaneous mutations occur at low frequency, as in the case of Mycobacterium tuberculosis where a minority population of organisms is resistant to isoniazid. In this situation the use of isoniazid alone will eventually result in overgrowth by this subpopulation of resistant organisms. Genetic resistance may be chromosomal or transferable on transposons or plasmids. Plasmidmediated resistance has been increasingly recognized among Gram-negative enteric pathogens. By the process of conjugation (Chapter 13), resistance plasmids may be transferred between bacteria of the same and different species and also different genera. Such resistance can code for multiple antibiotic resistance. For example, the penicillins, cephalosporins, chloramphenicol and the aminoglycosides are all subject to enzymatic inactivation, which may be plasmid-mediated. Knowledge of the local epidemiology of resistant pathogens within a hospital, and especially within high-dependency areas such as intensive care units, is invaluable in guiding appropriate drug selection. 2.4.1 Multidrug resistance In recent years multidrug resistance has increased 236
among certain pathogens. These include Staphylococcus aureus, enterococci and M. tuberculosis. Staph. aureus resistant to methicillin is known as methicillin-resistant Staph. aureus (MRSA). These strains are resistant to many antibiotics and have been responsible for major epidemics worldwide, usually in hospitals where they affect patients in high-dependency units such as intensive care units, burns units and cardiothoracic units. MRSA have the ability to colonize staff and patients and to spread readily among them. Several epidemic strains are currently circulating in the UK. The glycopeptides vancomycin or teicoplanin and the oxazolidinone linezolid are the currently recommended agents for treating patients infected with these organisms. Another serious resistance problem is that of drug-resistant enterococci. These include Enterococcus faecalis and, in particular, E. faecium. Resistance to the glycopeptides has again been a problem among patients in high-dependency units. Four different phenotypes are recognized (VanA, VanB, VanC and VanD). The VanA phenotype is resistant to both glycopeptides, while the others are sensitive to teicoplanin but demonstrate high (VanB) or intermediate (VanC) resistance to vancomycin; VanD resistance has only recently been described and remains uncommon. Those fully resistant to the glycopeptides are increasing in frequency and causing great concern as they are essentially resistant to almost all antibiotics. Tuberculosis is on the increase after decades in which the incidence had been steadily falling. Drug-resistant strains have emerged largely among inadequately treated or non-compliant patients. These include the homeless, alcoholic, intravenous drug abusing and immigrant populations. Resistance patterns vary but increasingly include rifampicin and isoniazid. Furthermore, outbreaks of multidrug-resistant tuberculosis have been increasingly reported from a number of hospital centres in the USA and more recently Europe, including the UK. These infections have occasionally spread to health-care workers and are giving rise to considerable concern. The underlying mechanisms of resistance are considered in Chapter 13.
Clinical uses of antimicrobial drugs
2.5 Drug combinations
2.6 Adverse reactions
Antibiotics are generally used alone, but may on occasion be prescribed in combination. Combining two antibiotics may result in synergism, indifference or antagonism. In the case of synergism, microbial inhibition is achieved at concentrations below that for each agent alone and may prove advantageous in treating relatively insusceptible infections such as enterococcal endocarditis, where a combination of penicillin and gentamicin is synergistically active. Another advantage of synergistic combinations is that it may enable the use of toxic agents where dose reductions are possible. For example, meningitis caused by the fungus Cryptococcus neoformans responds to an abbreviated course of amphotericin B when it is combined with 5-flucytosine, thereby reducing the risk of toxicity from amphotericin B. Combined drug use is occasionally recommended to prevent resistance emerging during treatment. For example, treatment may fail when fusidic acid is used alone to treat Staph. aureus infections, because resistant strains develop rapidly; this is prevented by combining fusidic acid with flucloxacillin. Likewise, tuberculosis is initially treated with a minimum of three agents, such as rifampicin, isoniazid and pyrazinamide; again drug resistance is prevented, which may result if either agent is used alone. The most common reason for using combined therapy is in the treatment of confirmed or suspected mixed infections where a single agent alone will fail to cover all pathogenic organisms. This is the case in serious abdominal sepsis where mixed aerobic and anaerobic infections are common and the use of metronidazole in combination with either an aminoglycoside or a broad-spectrum cephalosporin is essential. Finally, drugs are used in combination in patients who are seriously ill and about whom uncertainty exists concerning the microbiological nature of their infection. This initial ‘blind therapy’ frequently includes a broadspectrum penicillin or cephalosporin in combination with an aminoglycoside. The regimen should be modified in the light of subsequent microbiological information.
Regrettably, all chemotherapeutic agents have the potential to produce adverse reactions with varying degrees of frequency and severity, and these include hypersensitivity reactions and toxic effects. These may be dose-related and predictable in a patient with a history of hypersensitivity or a previous toxic reaction to a drug or its chemical analogues. However, many adverse events are idiosyncratic and therefore unpredictable. Hypersensitivity reactions range in severity from fatal anaphylaxis, in which there is widespread tissue oedema, airway obstruction and cardiovascular collapse, to minor and reversible hypersensitivity reactions such as skin eruptions and drug fever. Such reactions are more likely in those with a history of hypersensitivity to the drug, and are more frequent in patients with previous allergic diseases such as childhood eczema or asthma. It is important to question patients closely concerning hypersensitivity reactions before prescribing, as it precludes the use of all compounds within a class, such as the sulphonamides or tetracyclines, while cephalosporins should be used with caution in patients who are allergic to penicillin because these agents are structurally related. They should be avoided entirely in those who have had a previous severe hypersensitivity reaction to penicillin. Drug toxicity is often dose-related and may affect a variety of organs or tissues. For example, the aminoglycosides are both nephrotoxic and ototoxic to varying degrees; therefore, dosaging should be individualized and the serum assayed, especially where renal function is abnormal, to avoid toxic effects and non-therapeutic drug concentrations. An example of dose-related toxicity is chloramphenicol-induced bone marrow suppression. Chloramphenicol interferes with the normal maturation of bone marrow stem cells and high concentrations may result in a steady fall in circulating red and white cells and also platelets. This effect is generally reversible with dose reduction or drug withdrawal. This dose-related toxic reaction of chloramphenicol should be contrasted with idiosyncratic bone marrow toxicity which is unrelated to dose and occurs at a much lower frequency of approximately 1:40 000 and is frequently irreversible, 237
Chapter 14
ending fatally. Toxic effects may also be genetically determined. For example, peripheral neuropathy may occur in those who are slow acetylators of isoniazid, while haemolysis occurs in those deficient in the red cell enzyme glucose-6-phosphate dehydrogenase, when treated with sulphonamides or primaquine. 2.7 Superinfection Anti-infective drugs not only affect the invading organism undergoing treatment but also have an impact on the normal bacterial flora, especially of the skin and mucous membranes. This may result in microbial overgrowth of resistant organisms with subsequent superinfection. One example is the common occurrence of oral or vaginal candidiasis in patients treated with broad-spectrum agents such as ampicillin or tetracycline. A more serious example is the development of pseudomembranous colitis from the overgrowth of toxin-producing strains of Clostridium difficile present in the bowel flora following the use of clindamycin and other broad-spectrum antibiotics. This condition is managed by drug withdrawal and oral vancomycin. Rarely, colectomy (excision of part or whole of the colon) may be necessary for severe cases.
phylaxis be limited to the peri-operative period, the first dose being administered approximately 1 hour before surgery for injectable agents and repeated for a maximum of two to three repeat doses postoperatively. Prolonging chemoprophylaxis beyond this period is not cost-effective and increases the risk of adverse drug reactions and superinfection. One of the best examples of the efficacy of surgical prophylaxis is in the area of large bowel surgery. Before the widespread use of chemoprophylaxis, postoperative infection rates for colectomy were often 30% or higher; these have now been reduced to around 5%. Chemoprophylaxis has been extended to other surgical procedures where the risk of infection may be low but its occurrence has serious consequences. This is especially true for the implantation of prosthetic joints or heart valves. These are major surgical procedures and although infection may be infrequent its consequences are serious and on balance the use of chemoprophylaxis is cost-effective. Examples of chemoprophylaxis in the nonsurgical arena include the prevention of endocarditis with amoxicillin in patients with valvular heart disease undergoing dental surgery, and the prevention of secondary cases of meningococcal meningitis with rifampicin among household contacts of an index case.
2.8 Chemoprophylaxis An increasingly important use of antimicrobial agents is that of infection prevention, especially in relationship to surgery. Infection remains one of the most important complications of many surgical procedures, and the recognition that peri-operative antibiotics are effective and safe in preventing this complication has proved a major advance in surgery. The principles that underlie the chemoprophylactic use of antibacterials relate to the predictability of infection for a particular surgical procedure, both in terms of its occurrence, microbial aetiology and susceptibility to antibiotics. Therapeutic drug concentrations present at the operative site at the time of surgery rapidly reduce the number of potentially infectious organisms and prevent wound sepsis. If prophylaxis is delayed to the postoperative period then efficacy is markedly impaired. It is important that chemopro238
3 Clinical use The choice of antimicrobial chemotherapy is initially dependent on the clinical diagnosis. Under some circumstances the clinical diagnosis implies a microbiological diagnosis which may dictate specific therapy. For example, typhoid fever is caused by Salmonella typhi, which is generally sensitive to chloramphenicol, co-trimoxazole and ciprofloxacin. However, for many infections, establishing a clinical diagnosis implies a range of possible microbiological causes and requires laboratory confirmation from samples collected, preferably before antibiotic therapy is begun. Laboratory isolation and susceptibility testing of the causative agent establish the diagnosis with certainty and make drug selection more rational. However, in many circumstances, especially in general practice,
Clinical uses of antimicrobial drugs
microbiological documentation of an infection is not possible. Hence knowledge of the usual microbiological cause of a particular infection and its susceptibility to antimicrobial agents is essential for effective drug prescribing. The following section explores a selection of the problems associated with antimicrobial drug prescribing for a range of clinical conditions. 3.1 Respiratory tract infections Infections of the respiratory tract are among the commonest of infections, and account for much consultation in general practice and a high percentage of acute hospital admissions. They are divided into infections of the upper respiratory tract, involving the ears, throat, nasal sinuses and the trachea, and the lower respiratory tract (LRT), where they affect the airways, lungs and pleura. 3.1.1 Upper respiratory tract infections Acute pharyngitis presents a diagnostic and therapeutic dilemma. The majority of sore throats are caused by a variety of viruses; fewer than 20% are bacterial and hence potentially responsive to antibiotic therapy. However, antibiotics are widely prescribed and this reflects the difficulty in discriminating streptococcal from non-streptococcal infections clinically in the absence of microbiological documentation. Nonetheless, Strep. pyogenes is the most important bacterial pathogen and this responds to oral penicillin. However, up to 10 days’ treatment is required for its eradication from the throat. This requirement causes problems with compliance as symptomatic improvement generally occurs within 2–3 days. Although viral infections are important causes of both otitis media and sinusitis, they are generally self-limiting. Bacterial infections may complicate viral illnesses, and are also primary causes of ear and sinus infections. Streptococcus pneumoniae and Haemophilus influenzae are the commonest bacterial pathogens. Amoxicillin is widely prescribed for these infections as it is microbiologically active, penetrates the middle ear and sinuses, is well tolerated and has proved effective.
3.1.2 Lower respiratory tract infections Infections of the LRT include pneumonia, lung abscess, bronchitis, bronchiectasis and infective complications of cystic fibrosis. Each presents a specific diagnostic and therapeutic challenge, which reflects the variety of pathogens involved and the frequent difficulties in establishing an accurate microbial diagnosis. The laboratory diagnosis of LRT infections is largely dependent upon culturing sputum. Unfortunately this may be contaminated with the normal bacterial flora of the upper respiratory tract during expectoration. In hospitalized patients, the empirical use of antibiotics before admission substantially diminishes the value of sputum culture and may result in overgrowth by non-pathogenic microbes, thus causing difficulty with the interpretation of sputum culture results. Alternative diagnostic samples include needle aspiration of sputum directly from the trachea or of fluid within the pleural cavity. Blood may also be cultured and serum examined for antibody responses or microbial antigens. In the community, few patients will have their LRT infection diagnosed microbiologically and the choice of antibiotic is based on clinical diagnosis. Pneumonia. The range of pathogens causing acute pneumonia includes viruses, bacteria and, in the immunocompromised host, parasites and fungi. Table 14.2 summarizes these pathogens and indicates drugs appropriate for their treatment. Clinical assessment includes details of the evolution of the infection, any evidence of a recent viral infection, the age of the patient and risk factors such as corticosteroid therapy or pre-existing lung disease. The extent of the pneumonia, as assessed clinically or by X-ray, is also important. Streptococcus pneumoniae remains the commonest cause of pneumonia and still responds well to penicillin despite a global increase in isolates showing reduced susceptibility to this agent. In addition, a number of atypical infections may cause pneumonia and include Mycoplasma pneumoniae, Legionella pneumophila, psittacosis and occasionally Q fever. With psittacosis there may be a history of contact with parrots or budgerigars; while Legionnaires’ disease has often been acquired during hotel holidays in the Mediterranean area. The 239
Chapter 14 Table 14.2 Microorganisms responsible for pneumonia and the therapeutic agent of choice Pathogen
Drug(s) of choice
Streptococcus pneumoniae Staphylococcus aureus Haemophilus influenzae
Penicillin Flucloxacillin ± fusidic acid Cefotaxime or ciprofloxacin Cefotaxime ± gentamicin Gentamicin ± ceftazidime Erythromycin or tetracycline Erythromycin ± rifampicin Tetracycline Rifampicin + isoniazid + ethambutol + pyrazinamide* Aciclovir Fluconazole Amphotericin B Penicillin or metronidazole
Klebsiella pneumoniae Pseudomonas aeruginosa Mycoplasma pneumoniae Legionella pneumophila Chlamydia psittaci Mycobacterium tuberculosis
Herpes simplex, varicella/zoster Candida spp. Aspergillus spp. Anaerobic bacteria
* Reduce to two drugs after 6–8 weeks.
atypical pneumonias, unlike pneumococcal pneumonia, do not respond to penicillin. Legionnaires’ disease is treated with erythromycin and, in the presence of severe pneumonia, rifampicin is added to the regimen. Mycoplasma infections are best treated with either erythromycin or tetracycline, while the latter drug is indicated for both psittacosis and Q fever. Lung abscess. Destruction of lung tissue may lead to abscess formation and is a feature of aerobic Gram-negative bacillary and Staph. aureus infections. In addition, aspiration of oropharyngeal secretion can lead to chronic low-grade sepsis with abscess formation and the expectoration of foulsmelling sputum that characterizes anaerobic sepsis. The latter condition responds to high-dose penicillin, which is active against most of the normal oropharyngeal flora, while metronidazole may be appropriate for strictly anaerobic infections. In the case of aerobic Gram-negative bacillary sepsis, aminoglycosides, with or without a broadspectrum cephalosporin, are the agents of choice. Acute staphylococcal pneumonia is an extremely serious infection and requires treatment with highdose flucloxacillin alone or in combination with fusidic acid. 240
Cystic fibrosis. Cystic fibrosis is a multi-system, congenital abnormality that often affects the lungs and results in recurrent infections, initially with Staph. aureus, subsequently with H. influenzae and eventually leads on to recurrent Pseudomonas aeruginosa infection. The last organism is associated with copious quantities of purulent sputum that are extremely difficult to expectorate. Ps. aeruginosa is a co-factor in the progressive lung damage that is eventually fatal in these patients. Repeated courses of antibiotics are prescribed and although they have improved the quality and longevity of life, infections caused by Ps. aeruginosa are difficult to treat and require repeated hospitalization and administration of parenteral antibiotics such as an aminoglycoside, either alone or in combination with an antipseudomonal penicillin or cephalosporin. The dose of aminoglycosides tolerated by these patients is often higher than in normal individuals and is associated with larger volumes of distribution for these and other agents. Some benefit may also be obtained from inhaled aerosolized antibiotics. Unfortunately drug resistance may emerge and makes drug selection more dependent upon laboratory guidance. 3.2 Urinary tract infections Urinary tract infection is a common problem in both community and hospital practice. Although occurring throughout life, infections are more common in pre-school girls and women during their childbearing years, although in the elderly the sex distribution is similar. Infection is predisposed by factors that impair urine flow. These include congenital abnormalities, reflux of urine from the bladder into the ureters, kidney stones and tumours and, in males, enlargement of the prostate gland. Bladder catheterization is an important cause of urinary tract infection in hospitalized patients. 3.2.1 Pathogenesis In those with structural or drainage problems the risk exists of ascending infection to involve the kidney and occasionally the bloodstream. Although structural abnormalities may be absent in women of childbearing years, infection can become
Clinical uses of antimicrobial drugs
recurrent, symptomatic and extremely distressing. Of greater concern is the occurrence of infection in the pre-school child, as normal maturation of the kidney may be impaired and may result in progressive damage which presents as renal failure in later life. From a therapeutic point of view, it is essential to confirm the presence of bacteriuria (a condition in which there are bacteria in the urine), as symptoms alone are not a reliable method of documenting infection. This applies particularly to bladder infection, where the symptoms of burning micturition (dysuria) and frequency can be associated with a variety of non-bacteriuric conditions. Patients with symptomatic bacteriuria should always be treated. However, the necessity to treat asymptomatic bacteriuric patients varies with age and the presence or absence of underlying urinary tract abnormalities. In the pre-school child it is essential to treat all urinary tract infections and maintain the urine in a sterile state so that normal kidney maturation can proceed. Likewise in pregnancy there is a risk of infection ascending from the bladder to involve the kidney. This is a serious complication and may result in premature labour. Other indications for treating asymptomatic bacteriuria include the presence of underlying renal abnormalities such as stones, which may be associated with repeated infections caused by Proteus spp. 3.2.2 Drug therapy The antimicrobial treatment of urinary tract infection presents a number of interesting challenges. Drugs must be selected for their ability to achieve high urinary concentrations and, if the kidney is involved, adequate tissue concentrations. Safety in childhood or pregnancy is important as repeated or prolonged medication may be necessary. The choice of agent will be dictated by the microbial aetiology and susceptibility findings, because the latter can vary widely among Gram-negative enteric bacilli, especially in patients who are hospitalized. Table 14.3 shows the distribution of bacteria causing urinary tract infection in the community and in hospitalized patients. The greater tendency towards infections caused by Klebsiella spp. and Ps. aeruginosa should be noted as antibiotic sensitivity
Table 14.3 Urinary tract infection — distribution of pathogenic bacteria in the community and hospitalized patients Organism
Community (%)
Hospital (%)
Escherichia coli Proteus mirabilis Klebsiella or Enterobacter spp. Enterococci Staphylococcus epidermidis Pseudomonas aeruginosa
75 10 4 6 5 –
55 13 18 5 4 5
is more variable for these pathogens. Drug resistance has increased substantially in recent years and has reduced the value of formerly widely prescribed agents such as the sulphonamides and ampicillin. Uncomplicated community-acquired urinary tract infection presents few problems with management. Drugs such as trimethoprim, ciprofloxacin and ampicillin are widely used. Cure rates are close to 100% for ciprofloxacin, about 80% for trimethoprim and about 50% for ampicillin — to which resistance has been steadily increasing. Treatment for 3 days is generally satisfactory and is usually accompanied by prompt control of symptoms. Single-dose therapy with amoxicillin 3 g has also been shown to be effective in selected individuals. Alternative agents include nitrofurantoin, nalidixic acid and norfloxacin, although these are not as well tolerated. Oral cephalosporins and co-amoxiclav are also used. It is important to demonstrate the cure of bacteriuria with a repeat urine sample collected 4–6 weeks after treatment, or sooner should symptoms fail to subside. Recurrent urinary tract infection is an indication for further investigation of the urinary tract to detect underlying pathology that may be surgically correctable. Under these circumstances it also is important to maintain the urine in a sterile state. This can be achieved with repeated courses of antibiotics, guided by laboratory sensitivity data. Alternatively, long-term chemoprophylaxis for periods of 6 months to control infection by either prevention or suppression is widely used. Trimethoprim is the most commonly prescribed chemoprophylactic agent and is given as a single nightly dose. This achieves high urinary concentra241
Chapter 14
tions throughout the night and generally ensures a sterile urine. Nitrofurantoin is an alternative agent. Infection of the kidney demands the use of agents that achieve adequate tissue as well as urinary concentrations. As bacteraemia (a condition in which there are bacteria circulating in the blood) may complicate infection of the kidney, it is generally recommended that antibiotics be administered parenterally. Although ampicillin was formerly widely used, drug resistance is now common and agents such as cefotaxime or ciprofloxacin are often preferred, because the aminoglycosides, although highly effective and preferentially concentrated within the renal cortex, carry the risk of nephrotoxicity. Infections of the prostate tend to be persistent, recurrent and difficult to treat. This is in part due to the more acid environment of the prostate gland, which inhibits drug penetration by many of the antibiotics used to treat urinary tract infection. Agents that are basic in nature, such as erythromycin, achieve therapeutic concentrations within the gland but unfortunately are not active against the pathogens responsible for bacterial prostatitis. Trimethoprim, however, is a useful agent as it is preferentially concentrated within the prostate and is active against many of the causative pathogens. It is important that treatment be prolonged for several weeks, as relapse is common. 3.3 Gastrointestinal infections The gut is vulnerable to infection by viruses, bacteria, parasites and occasionally fungi. Virus infections are the most prevalent but are not susceptible to chemotherapeutic intervention. Bacterial infections are more readily recognized and raise questions concerning the role of antibiotic management. Parasitic infections of the gut are beyond the scope of this chapter. Bacteria cause disease of the gut as a result of either mucosal invasion or toxin production or a combination of the two mechanisms, as summarized in Table 14.4. Treatment is largely directed at replacing and maintaining an adequate intake of fluid and electrolytes. Antibiotics are generally not recommended for infective gastroenteritis, but deserve consideration where they have been 242
Table 14.4 Bacterial gut infections — pathogenic mechanisms Origin
Site of infection Mechanism
Campylobacter jejuni
Small and large bowel Small and large bowel Large bowel
Salmonella spp. Shigella spp. Escherichia coli enteroinvasive enterotoxigenic Clostridium difficile Staphylococcus aureus Vibrio cholerae Clostridium perfringens Yersinia spp. Bacillus cereus Vibrio parahaemolyticus
Large bowel Small bowel Large bowel Small bowel Small bowel Small bowel Small and large bowel Small bowel Small bowel
Invasion Invasion Invasion ± toxin Invasion Toxin Toxin Toxin Toxin Toxin Invasion Invasion ± toxin Invasion + toxin
demonstrated to abbreviate the acute disease or to prevent complications including prolonged gastrointestinal excretion of the pathogen where this poses a public health hazard. It should be emphasized that most gut infections are self-limiting. However, attacks can be severe and may result in hospitalization. Antibiotics are used to treat severe Campylobacter and Shigella infections; erythromycin and ciprofloxacin, respectively, are the preferred agents. Such treatment abbreviates the disease and eliminates gut excretion in Shigella infection. However, in severe Campylobacter infection the data are currently equivocal, although the clinical impression favours the use of erythromycin for severe infections. The role of antibiotics for Campylobacter and Shigella infections should be contrasted with gastrointestinal salmonellosis, for which antibiotics are contraindicated as they do not abbreviate symptoms, are associated with more prolonged gut excretion and introduce the risk of adverse drug reactions. However, in severe salmonellosis, especially at extremes of age, systemic toxaemia and bloodstream infection can occur and under these circumstances treatment with either ciprofloxacin or trimethoprim is appropriate.
Clinical uses of antimicrobial drugs
Typhoid and paratyphoid fevers (known as enteric fevers), although acquired by ingestion of salmonellae, Sal. typhi and Sal. paratyphi, respectively, are largely systemic infections and antibiotic therapy is mandatory; ciprofloxacin is now the drug of choice although trimethoprim or chloramphenicol are satisfactory alternatives. Prolonged gut excretion of Sal. typhi is a well-known complication of typhoid fever and is a major public health hazard in developing countries. Treatment with ciprofloxacin or high dose ampicillin can eliminate the gall bladder excretion which is the major site of persistent infection in carriers. However, the presence of gallstones reduces the chance of cure. Cholera is a serious infection causing epidemics throughout Asia. Although a toxin-mediated disease, largely controlled with replacement of fluid and electrolyte losses, tetracycline has proved effective in eliminating the causative vibrio from the bowel, thereby abbreviating the course of the illness and reducing the total fluid and electrolyte losses. Traveller’s diarrhoea may be caused by one of many gastrointestinal pathogens (Table 14.4). However, enterotoxigenic Escherichia coli is the most common pathogen. While it is generally shortlived, traveller’s diarrhoea can seriously mar a brief period abroad, be it for holiday or business purposes. Although not universally accepted, the use of short-course trimethoprim or quinolone such as norfloxacin can abbreviate an attack in patients with severe disease. 3.4 Skin and soft tissue infections Infections of the skin and soft tissue commonly follow traumatic injury to the epithelium but occasionally may be bloodborne. Interruption of the integrity of the skin allows ingress of microorganisms to produce superficial, localized infections which on occasion may become more deepseated and spread rapidly through tissues. Skin trauma complicates surgical incisions and accidents, including burns. Similarly, prolonged immobilization can result in pressure damage to skin from impaired blood flow. It is most commonly seen in patients who are unconscious. Microbes responsible for skin infection often arise from the normal skin flora, which includes
Staph. aureus. In addition Strep. pyogenes, Ps. aeruginosa and anaerobic bacteria are other recognized pathogens. Viruses also affect the skin and mucosal surfaces, either as a result of generalized infection or localized disease as in the case of herpes simplex. The latter is amenable to antiviral therapy in selected patients, although for the majority of patients, virus infections of the skin are self-limiting. Strep. pyogenes is responsible for a range of skin infections: impetigo is a superficial infection of the epidermis which is common in childhood and is highly contagious; cellulitis is a more deep-seated infection which spreads rapidly through the tissues to involve the lymphatics and occasionally the bloodstream; erysipelas is a rapidly spreading cellulitis commonly involving the face, which characteristically has a raised leading edge due to lymphatic involvement. Necrotizing fasciitis is a more serious, rapidly progressive infection of the skin and subcutaneous structures including the fascia and musculature. Despite early diagnosis and high-dose intravenous antibiotics, this condition is often life-threatening and may require extensive surgical debridement of devitalized tissue and even limb amputation to ensure survival. A fatal outcome is usually the result of profound toxaemia and bloodstream spread. Penicillin is the drug of choice for all these infections although in severe instances parenteral administration is appropriate. The use of topical agents, such as tetracycline, to treat impetigo may fail as drug resistance is now recognized. Staph. aureus is responsible for a variety of skin infections which require therapeutic approaches different from those of streptococcal infections. Staphylococcal cellulitis is indistinguishable clinically from streptococcal cellulitis and responds to flucloxacillin, but generally fails to respond to penicillin owing to penicillinase (b-lactamase) production. Staph. aureus is an important cause of superficial, localized skin sepsis which varies from small pustules to boils and occasionally to a more deeply invasive, suppurative skin abscess known as a carbuncle. Antibiotics are generally not indicated for these conditions. Pustules and boils settle with antiseptic soaps or creams and often discharge spontaneously, whereas carbuncles frequently require surgical drainage. Staph. aureus may also 243
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cause postoperative wound infections, sometimes associated with retained suture material, and settles once the stitch is removed. Antibiotics are only appropriate in this situation if there is extensive accompanying soft tissue invasion. Anaerobic bacteria are characteristically associated with foul-smelling wounds. They are found in association with surgical incisions following intra-abdominal procedures and pressure sores, which are usually located over the buttocks and hips where they become infected with faecal flora. These infections are frequently mixed and include Gram-negative enteric bacilli, which may mask the presence of underlying anaerobic bacteria. The principles of treating anaerobic soft tissue infection again emphasize the need for removal of all foreign and devitalized material. Antibiotics such as metronidazole or clindamycin should be considered where tissue invasion has occurred. The treatment of infected burn wounds presents a number of peculiar facets. Burns are initially sterile, especially when they involve all layers of the skin. However, they rapidly become colonized with bacteria whose growth is supported by the proteinrich exudate. Staphylococci, Strep. pyogenes and, particularly, Ps. aeruginosa frequently colonize burns and may jeopardize survival of skin grafts and occasionally, and more seriously, result in bloodstream invasion. Treatment of invasive Ps. aeruginosa infections requires combined therapy with an aminoglycoside, such as gentamicin or tobramycin, and an antipseudomonal agent, such as ceftazidime or piperacillin. This produces high therapeutic concentrations which generally act in a synergistic manner. The use of aminoglycosides in patients with serious burns requires careful monitoring of serum concentrations to ensure that they are therapeutic yet non-toxic, as renal function is often impaired in the days immediately following a serious burn. Excessive sodium loading may complicate the use of large doses of antipseudomonal penicillins such as piperacillin. 3.5 Central nervous system infections The brain, its surrounding covering of meninges and the spinal cord are subject to infection, which is generally bloodborne but may also complicate neu244
rosurgery, penetrating injuries or direct spread from infection in the middle ear or nasal sinuses. Viral meningitis is the most common infection but is generally self-limiting. Occasionally destructive forms of encephalitis occur; an example is herpes simplex encephalitis. Bacterial infections include meningitis and brain abscesses and carry a high risk of mortality, while in those who recover, residual neurological damage or impairment of intellectual function may follow. This occurs despite the availability of antibiotics active against the responsible bacterial pathogens. Fungal infections of the brain, although rare, are increasing in frequency, particularly among immunocompromised patients who either have underlying malignant conditions or are on potent cytotoxic drugs. The treatment of bacterial infections of the central nervous system highlights a number of important therapeutic considerations. Bacterial meningitis is caused by a variety of bacteria although their incidence varies with age. In the neonate, E. coli and group B streptococci account for the majority of infections, while in the preschool child H. influenzae was the commonest pathogen before the introduction of a highly effective vaccine. Neisseria meningitidis has a peak incidence between 5 and 15 years of age, while pneumococcal meningitis is predominantly a disease of adults. Penicillin is the drug of choice for the treatment of group B streptococcal, meningococcal and pneumococcal infections but, as discussed earlier, CSF concentrations of penicillin are significantly influenced by the intensity of the inflammatory response. To achieve therapeutic concentrations within the CSF, high dosages are required, and in the case of pneumococcal meningitis should be continued for 10–14 days. Resistance among Strep. pneumoniae to penicillin has increased worldwide. When causing meningitis, treatment is increasingly unsuccessful. Alternative agents include ceftriaxone and meropenem. Resistance of H. influenzae to ampicillin has increased in the past two decades and varies geographically. Thus, it can no longer be prescribed with confidence as initial therapy, and cefotaxime or ceftriaxone are now the preferred alternatives. However, once laboratory evidence for b-lactamase
Clinical uses of antimicrobial drugs
activity is excluded, ampicillin can be safely substituted. E. coli meningitis carries a mortality of greater than 40% and reflects both the virulence of this organism and the pharmacokinetic problems of achieving adequate CSF antibiotic levels. The broad-spectrum cephalosporins such as cefotaxime, ceftriaxone or ceftazidime have been shown to achieve satisfactory therapeutic levels and are the agents of choice to treat Gram-negative bacillary meningitis. Treatment again must be prolonged for periods ranging from 2 to 4 weeks. Brain abscess presents a different therapeutic challenge. An abscess is locally destructive to the brain and causes further damage by increasing intracranial pressure. The infecting organisms are varied but those arising from middle ear or nasal sinus infection are often polymicrobial and include anaerobic bacteria, micro-aerophilic species and Gram-negative enteric bacilli. Less commonly, a pure Staph. aureus abscess may complicate bloodborne spread. Brain abscess is a neurosurgical emergency and requires drainage. However, antibiotics are an important adjunct to treatment. The polymicrobial nature of many infections demands prompt and careful laboratory examination to determine optimum therapy. Drugs are selected not only on their ability to penetrate the blood–brain barrier and enter the CSF but also on their ability to penetrate the brain substance. Metronidazole has proved a valuable alternative agent in such infections, although it is not active against microaerophilic streptococci, which must be treated with high-dose benzylpenicillin. The two are often used in combination. Chloramphenicol is an alternative agent. 3.6 Fungal infections Fungal infections are divided into superficial or deep-seated infections. Superficial infections affect the skin, nails or mucosal surfaces of the mouth or genital tract. In contrast, deep-seated fungal diseases may target the lung or disseminate via the bloodstream to organs such as the brain, spleen, liver or skeletal system. The fungal infections of the skin and nails include Tinea pedis (athlete’s foot), T. capitis and T.
Table 14.5 Treatment recommendations for selected deep-seated fungal infections
Infection Candida spp. Cryptococcus neoformans Aspergillus spp. Mucormycosis
Preferred treatment
Alternative treatment
Fluconazole Fluconazole
Amphotericin B Amphotericin B ± flucytosine Itraconazole –
Amphotericin B Amphotericin B
carporis (ringworm), Candida intertrigo (usually groin and submammary regions) and pityriasis (Malassezia). A variety of topical and systemic antifungal agents are available. The imidazole class of drugs includes clotrimazole and miconazole, which are highly effective topically. Systemic antifungals used to treat superficial fungal infections include griseofulvin and terbinafine, which is an allylamine. Both agents are ineffective in the treatment of deep-seated fungal infections that may be caused by yeasts (Cryptococcus neoformans), yeast-like fungi (Candida spp.) or the filamentous fungi (Aspergillus spp). These produce a variety of syndromes for which different antifungal agents are indicated (Table 14.5). The polyenes include amphotericin B, which after many years remains the agent of choice for the treatment of a wide variety of life-threatening fungal diseases which often complicate cancer chemotherapy, organ transplantation and immunodeficiency diseases, such as AIDS. Nephrotoxicity is common but can be avoided by careful dosaging or the use of liposomal formulations. The other major class of systemic antifungals is the triazoles, which include fluconazole and itraconazole. These are extremely well tolerated but may interact with a number of drugs and drug classes such as the sulphonylureas, antihistamines and lipid-lowering agents among others. 3.7 Medical device-associated infections A wide variety of medical devices are increasingly used in clinical practice. These range from vasculature and urinary catheters, prosthetic joints and heart valves, shunts and stents for improving the flow of CSF, blood or bile according to their site of 245
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use, to intracardiac patches and vascular pumps. Unfortunately infection is the most frequent complication of their use and may result in the need to replace or remove the device, sometimes with potentially life-threatening and fatal consequences. Infections are often caused by organisms arising from the normal skin flora, which gain access at the time of insertion of the device. Staph. epidermidis is among the most frequent of isolates. Following attachment to the surface of the device, the organisms undergo multiplication with the formation of extracellular polysaccharide material (glycocalyx) which contains slowly replicating cells to form a biofilm. Microorganisms within a biofilm are less vulnerable to attack by host defences (phagocytes, complement and antibodies) and are relatively insusceptible to antibiotic therapy despite the variable ability of drugs to penetrate the biofilm. Management approaches have therefore emphasized the need for prevention through the addition of good sterile technique at the time of insertion. Manufacturers have also responded by using materials and creating surface characteristics of implanted materials inclement to microbial attachment. Likewise the use of prophylactic antibiotics at the time of insertion of deep-seated devices such as joint and heart valve prostheses has further reduced the risk of infection. Once a medical device becomes infected, management is difficult. Treatment with agents such as flucloxacillin, vancomycin and most recently linezolid is often unsuccessful and the only course of action is to remove the device.
4 Antibiotic policies 4.1 Rationale The plethora of available antimicrobial agents presents both an increasing problem of selection to the prescriber and difficulties for the diagnostic laboratory as to which agents should be tested for susceptibility. Differences in antimicrobial activity among related compounds are often of minor importance but can occasionally be of greater significance and may be a source of confusion to the non-specialist. This applies particularly to large classes of drugs, 246
such as the penicillins and cephalosporins, where there has been an explosion in the availability of new agents in recent years. Guidance, in the form of an antibiotic policy, has a major role to play in providing the prescriber with a range of agents appropriate to his/her needs and should be supported by laboratory evidence of susceptibility to these agents. In recent years, increased awareness of the cost of medical care has led to a major review of various aspects of health costs. The pharmacy budget has often attracted attention as, unlike many other hospital expenses, it is readily identifiable in terms of cost and prescriber. Thus, an antibiotic policy is also seen as a means whereby the economic burden of drug prescribing can be reduced or contained. There can be little argument with the recommendation that the cheaper of two compounds should be selected where agents are similar in terms of efficacy and adverse reactions. Likewise, generic substitution is also desirable provided that there is bioequivalence. It has become increasingly impractical for pharmacists to stock all the formulations of every antibiotic currently available, and here again an antibiotic policy can produce significant savings by limiting the amount of stock held. A policy based on a restricted number of agents also enables price reduction on purchasing costs through competitive tendering. The above activities have had a major influence on containing or reducing drug costs, although these savings have often been lost as new and often expensive preparations become available, particularly in the field of biological and anticancer therapy. Another increasingly important argument in favour of an antibiotic policy is the occurrence of drug-resistant bacteria within an institution. The presence of sick patients and the opportunities for the spread of microorganisms can produce outbreaks of hospital infection. The excessive use of selected agents has been associated with the emergence of drug-resistant bacteria which have often caused serious problems within highdependency areas, such as intensive care units or burns units where antibiotic use is often high. One oft-quoted example is the occurrence of a multiple antibiotic-resistant K. aerogenes within a neurosurgical intensive care unit in which the organism
Clinical uses of antimicrobial drugs
became resistant to all currently available antibiotics and was associated with the widespread use of ampicillin. By prohibiting the use of all antibiotics, and in particular ampicillin, the resistant organism rapidly disappeared and the problem was resolved. Currently the most important hospital-acquired pathogen is methicillin-resistant Staph. aureus, which is responsible for a range of serious infections such as pneumonia, postoperative wound infection and skin infections which may in turn be complicated by bloodstream spread. The use of vancomycin and teicoplanin has escalated as a consequence, and in turn has been linked to the emergence of vancomycin-resistant enterococci. In formulating an antibiotic policy, it is important that the susceptibility of microorganisms be monitored and reviewed at regular intervals. This applies not only to the hospital as a whole, but to specific high-dependency units in particular. Likewise general practitioner samples should also be monitored. This will provide accurate information on drug susceptibility to guide the prescriber as to the most effective agent.
4.2.2 Restricted reporting Another approach that is widely practised in the UK is that of restricted reporting. The laboratory, largely for practical reasons, tests only a limited range of agents against bacterial isolates. The agents may be selected primarily by microbiological staff or following consultation with their clinical colleagues. The antibiotics tested will vary according to the site of infection, as drugs used to treat urinary tract infections often differ from those used to treat systemic disease. There are specific problems regarding the testing of certain agents such as the cephalosporins, where the many different preparations have varying activity against bacteria. The practice of testing a single agent to represent first-generation, secondgeneration or third-generation compounds is questionable, and with the new compounds susceptibility should be tested specifically to that agent. By selecting a limited range of compounds for use, sensitivity testing becomes a practical consideration and allows the clinician to use such agents with greater confidence.
4.2 Types of antibiotic policies There are a number of different approaches to the organization of an antibiotic policy. These range from a deliberate absence of any restriction on prescribing to a strict policy whereby all anti-infective agents must have expert approval before they are administered. Restrictive policies vary according to whether they are mainly laboratory-controlled, by employing restrictive reporting, or whether they are mainly pharmacy-controlled, by restrictive dispensing. In many institutions it is common practice to combine the two approaches. 4.2.1 Free prescribing policy The advocates of a free prescribing policy argue that strict antibiotic policies are both impractical and limit clinical freedom to prescribe. It is also argued that the greater the number of agents in use the less likely it is that drug resistance will emerge to any one agent or class of agents. However, few would support such an approach, which is generally an argument for mayhem.
4.2.3 Restricted dispensing As mentioned above, the most Draconian of all antibiotic policies is the absolute restriction of drug dispensing pending expert approval. The expert opinion may be provided by either a microbiologist or infectious disease specialist. Such a system can only be effective in large institutions where staff are available 24 hours a day. This approach is often cumbersome, generates hostility and does not necessarily create the best educational forum for learning effective antibiotic prescribing. A more widely used approach is to divide agents into those approved for unrestricted use and those for restricted use. Agents on the unrestricted list are appropriate for the majority of common clinical situations. The restricted list may include agents where microbiological sensitivity information is essential, such as for vancomycin and certain aminoglycosides. In addition, agents that are used infrequently but for specific indications, such as parenteral amphotericin B, are also restricted in use. Other compounds that may be expensive and 247
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used for specific indications, such as broadspectrum b-lactams in the treatment of Ps. aeruginosa infections, may also be justifiably included on the restricted list. Items omitted from the restricted or unrestricted list are generally not stocked, although they can be obtained at short notice as necessary. Such a policy should have a mechanism whereby desirable new agents are added as they become available and is most appropriately decided at a therapeutics committee. Policing such a policy is best effected as a joint arrangement between senior pharmacists and microbiologists. This combined approach of both restricted reporting and restricted prescribing is extremely effective and provides a powerful educational tool for medical staff and students faced with learning the complexities of modern antibiotic prescribing. In some hospitals
248
‘Antibiotic Teams’ have emerged to advise and educate staff while monitoring compliance with prescribing policies as well as ensuring good standards of patient management.
5 Further reading Cohen, J. & Powderley, D. (2003) Infectious Diseases, 2nd edn. Mosby, Philadelphia. Finch, R. G. (2001) Antimicrobial therapy: principles of use. Medicine, 29, 35–40. Finch, R. G., Greenwood, D., Norrby, R. & Whitley, R. (2002) Antibiotic and Chemotherapy, 8th edn. Churchill Livingstone, Edinburgh. Greenwood, D. (2000) Antimicrobial Chemotherapy, 4th edn. Oxford University Press, Oxford. Mandell, G. L., Douglas, R. G. & Bennett, J. E. (2000) Principles and Practice of Infectious Diseases, 5th edn. Churchill Livingstone, Philadelphia.
Part 3 Microbiological Aspects of Pharmaceutical Processing
Chapter 15
Ecology of microorganisms as it affects the pharmaceutical industry Elaine Underwood 1 Introduction 2 Atmosphere 2.1 Microbial content 2.2 Reduction of microbial count 2.3 Compressed air 3 Water 3.1 Raw or mains water 3.2 Softened water 3.3 Deionized or demineralized water 3.4 Distilled water 3.5 Water produced by reverse osmosis 3.6 Distribution system 3.7 Disinfection of water 3.7.1 Chemical treatment 3.7.2 Filtration 3.7.3 Light 3.7.4 Microbial checks
1 Introduction The microbiological quality of pharmaceutical products is influenced by the environment in which they are manufactured and by the materials used in their formulation. With the exception of preparations which are terminally sterilized in their final container, the microflora of the final product may represent the contaminants from the raw materials, from the equipment with which it was made, from the atmosphere, from the person operating the process or from the final container into which it was packed. Some of the contaminants may be pathogenic while others may grow even in the presence of preservatives and spoil the product. Any microorganisms that are destroyed by in-process heat treatment may still leave cell residues which may be toxic or pyrogenic (Chapter 3), as the pyrogenic fraction, lipid A, which is present in the cell wall is not destroyed under the same conditions as the organisms.
4 Skin and respiratory tract flora 4.1 Microbial transfer from operators 4.2 Hygiene and protective clothing 5 Raw materials 6 Packaging 7 Buildings 7.1 Walls and ceilings 7.2 Floors and drains 7.3 Doors, windows and fittings 8 Equipment 8.1 Pipelines 8.2 Cleansing 8.3 Disinfection and sterilization 8.4 Microbial checks 9 Cleaning equipment and utensils 10 Further reading
In parallel with improvements in manufacturing technology there have been developments in Good Manufacturing Practices to minimize contamination by a study of the ecology of microorganisms, the hazards posed by them and any points in the process which are critical to their control. This approach has been distilled into the concept of Hazard Analysis of Critical Control Points (HACCP), with the objective of improving the microbiological safety of the product in a cost-effective manner, which has been assisted by the development of rapid methods for the detection of microorganisms.
2 Atmosphere 2.1 Microbial content Air is not a natural environment for the growth and reproduction of microorganisms, as it does not contain the necessary amount of moisture and nutrients 251
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in a form that can be utilized. However, almost any sample of untreated air contains suspended bacteria, moulds and yeasts, but to survive they must be able to tolerate desiccation and the continuing dry state. Microorganisms commonly isolated from air are the spore-forming bacteria Bacillus spp. and Clostridium spp., the non-sporing bacteria Staphylococcus spp., Streptococcus spp. and Corynebacterium spp., the moulds Penicillium spp., Cladosporium spp., Aspergillus spp. and Mucor spp., as well as the yeast Rhodotorula spp. The number of organisms in the atmosphere depends on the activity in the environment and the amount of dust that is disturbed. An area containing working machinery and active personnel will have a higher microbial count than one with a still atmosphere, and the air count of a dirty, untidy room will be greater than that of a clean room. The microbial air count is also influenced by humidity. A damp atmosphere usually contains fewer organisms than a dry one, as the contaminants are carried down by the droplets of moisture. Thus, the air in a cold store is usually free from microorganisms and air is less contaminated during the wet winter months than in the drier summer months. Microorganisms are carried into the atmosphere suspended on particles of dust, skin or clothing, or in droplets of moisture or sputum following talking, coughing or sneezing. The size of the particles to which the organisms are attached, together with the humidity of the air, determines the rate at which they will settle out. Bacteria and moulds not attached to suspended matter will settle out slowly in a quiet atmosphere. The rate of settling out will depend upon air current caused by ventilation, air extraction systems, convection currents above heat sources and the activity in the room. The microbial content of the air may be increased during the handling of contaminated materials during dispensing, blending and their addition to formulations. In particular, the use of starches and some sugars in the dry state may increase the mould count. Some packaging components, e.g. card and paperboard, have a microflora of both moulds and bacteria, and this is often reflected in high counts around packaging machines. Common methods for checking the microbiological quality of air include the following: 252
1 The exposure of Petri dishes containing a nutrient agar to the atmosphere for a given length of time. This relies upon microorganisms or dust particles bearing them settling on the surface. 2 The use of an air-sampling machine which draws a measured volume of air from the environment and impinges it on a nutrient agar surface on either a Petri dish, a plastic strip or a membrane filter which may then be incubated with a nutrient medium. This method provides valuable information in areas of low microbial contamination, particularly if the sample is taken close to the working area. The type of formulation being prepared determines the microbiological standard of the air supply required and the hazard it poses. In areas where products for injection and ophthalmic use which cannot be terminally sterilized by moist heat are being manufactured, the air count should be very low and regarded as a critical control point in the process, as although these products are required to pass a test for sterility (Chapter 20), the test itself is destructive, and therefore only relatively few samples are tested. An unsatisfactory air count may lead to the casual contamination of a few containers and may be undetected by the test for sterility. In addition, if the microbiological air quality is identified as a critical point, it may also give an early warning of potential contamination and permit timely correction. The manufacture of liquid or semi-solid preparations for either oral or topical use requires a clean environment for both the production and filling stages. While many formulations are adequately protected by chemical preservatives or a pH unfavourable to airborne bacteria that may settle in them, preservation against mould spores is more difficult to achieve. 2.2 Reduction of microbial count The microbial count of air may be reduced by filtration, chemical disinfection and to a limited extent by ultraviolet (UV) light. Filtration is the most commonly used method and filters may be made of a variety of materials such as cellulose, glass wool, fibreglass mixtures or polytetrafluorethylene (PTFE) with resin or acrylic binders. There are standards in both the UK and USA for the quality of moving air, in the UK there is a grading system from
Ecology of microorganisms as it affects the pharmaceutical industry
A to D and in the USA, six classes from class 1 to class 100 000. For the most critical aseptic work, it may be necessary to remove all particles in excess of 0.1 mm in size using a high efficiency particulate air (HEPA) filter, but for many operations a standard of < 100 particles per 3.5 litres (1.0 ft3) of 0.5 mm or larger (grade A in the UK — class 100 in the USA) is adequate. Such fine filtration is usually preceded by a coarse filter stage, or any suspended matter is removed by passing the air through an electrostatic field. To maintain efficiency, all air filters must be kept dry, as microorganisms may be capable of movement along continuous wet films and may be carried through a damp filter. Filtered air may be used to purge a complete room, or it may be confined to a specific area and incorporate the principle of laminar flow, which permits operations to be carried out in a gentle current of sterile air. The direction of the airflow may be horizontal or vertical, depending on the type of equipment being used, the type of operation and the material being handled. It is important that there is no obstruction between the air supply and the exposed product, as this may result in the deflection of microorganisms or particulate matter from a non-sterile surface and cause contamination. Airflow gauges are essential to monitor that the correct flow rate is obtained in laminar flow units and in complete suites to ensure that a positive pressure from clean to less clean areas is always maintained. The integrity of the air-filtration system must be checked regularly, and the most common method is by counting the particulate matter both in the working area and across the surface of the filter. For systems which have complex ducting or where the surfaces of the terminal filters are recessed, smoke tests using a chemical of known particulate size may be introduced just after the main fan and monitored at each outlet. The test has a twofold application, as both the terminal filter and any leaks in the ducting can be checked. These methods are useful in conjunction with those for determining the microbial air count as given earlier. Chemical disinfectants are limited in their use as air sterilants because of their irritant properties when sprayed. However, some success has been achieved with atomized propylene glycol at a con-
centration of 0.05–0.5 mg/L and quaternary ammonium compounds (QACs) at 0.075% may be used. For areas that can be effectively sealed off for fumigation purposes, formaldehyde gas at a concentration of 1–2 mg/L of air at a relative humidity of 80–90% is effective. UV irradiation at wavelengths between 240 and 280 nm (2400 and 2800 Å) is used to reduce bacterial contamination of air, but it is only active at a relatively short distance from the source. Bacteria and mould spores, particularly those with heavily pigmented spore coats, are often resistant to such treatment. It is however, useful if used in combination with air filtration. 2.3 Compressed air Compressed air has many applications in the manufacture of pharmaceutical products. A few examples of its uses are the conveyance of powders and suspension, providing aeration for some fermentations and as a power supply for the reduction of particle size by impaction. Unless it is sterilized by filtration or a combination of heat and filtration, microorganisms present will be introduced into the product. The microbial content of compressed air may be assessed by bubbling a known volume through a nutrient liquid and either filtering through a membrane, which is then incubated with a nutrient agar and a total viable count made, or the microbial content may be estimated more rapidly using techniques developed to detect changes in physical or chemical characteristics in the nutrient liquid.
3 Water The microbial ecology of water is of great importance in the pharmaceutical industry owing to its multiple uses as a constituent of many products as well as for various washing and cooling processes. Two main aspects are involved: the quality of the raw water and any processing it receives and the distribution system. Both should be taken into consideration when reviewing the hazards to the finished product and any critical control points. Microorganisms indigenous to fresh water in253
Chapter 15
clude Pseudomonas spp., Alcaligenes spp., Flavobacterium spp., Chromobacter spp. and Serratia spp. Such bacteria are nutritionally undemanding and often have a relatively low optimum growth temperature. Bacteria which are introduced as a result of soil erosion, heavy rainfall and decaying plant matter include Bacillus subtilis, B. megaterium, Enterobacter aerogenes and Enterobacter cloacae. Contamination by sewage results in the presence of Proteus spp., Escherichia coli and other enterobacteria, Streptococcus faecalis and Clostridium spp. Bacteria which are introduced as a result of animal or plant debris usually die as a result of the unfavourable conditions. An examination of stored industrial water supplies showed that 98% of the contaminants were Gram-negative bacteria; other organisms isolated were Micrococcus spp., Cytophaga spp., yeast, yeast-like fungi and actinomycetes. 3.1 Raw or mains water The quality of the water from the mains supply varies with both the source and the local authority, and while it is free from known pathogens and from faecal contaminants such as E. coli, it may contain other microorganisms. When the supply is derived from surface water the flora is usually more abundant and faster-growing than that of supplies from a deep-water source such as a well or spring. This is due to surface waters receiving both microorganisms and nutrients from soil and sewage while water from deep sources has its microflora filtered out. On prolonged storage in a reservoir, waterborne organisms tend to settle out, but in industrial storage tanks the intermittent throughput ensures that, unless treated, the contents of the tank serve as a source of infection. The bacterial count may rise rapidly in such tanks during summer months and reach 105–106 per ml. One of the uses of mains water is for washing chemicals used in pharmaceutical preparations to remove impurities or unwanted by-products of a reaction, and although the bacterial count of the water may be low, the volume used is large and the material being washed may be exposed to a considerable number of bacteria. The microbial count of the mains water will be re254
flected in both softened and deionized water which may be prepared from it. 3.2 Softened water This is usually prepared by either a base-exchange method using sodium zeolite, by a lime-soda ash process, or by the addition of sodium hexametaphosphate. In addition to the bacteria derived from the mains water, additional flora of Bacillus spp. and Staphylococcus aureus may be introduced into systems which use brine for regeneration and from the chemical filter beds which, unless treated, can act as a reservoir for bacteria. Softened water is often used for washing containers before filling with liquid or semi-solid preparations and for cooling systems. Unless precautions are taken, the microbial count in a cooling system or jacketed vessel will rise rapidly and if faults develop in the cooling plates or vessel wall, contamination of the product may occur. 3.3 Deionized or demineralized water Deionized water is prepared by passing mains water through anion and cation exchange resin beds to remove the ions. Thus, any bacteria present in the mains water will also be present in the deionized water, and beds which are not regenerated frequently with strong acid or alkali are often heavily contaminated and add to the bacterial content of the water. This problem has prompted the development of resins able to resist microbiological contamination. One such resin, a large-pore, strong-base, macroreticular, quaternary ammonium anion exchange resin which permits microorganisms to enter the pore cavity and then electrostatically binds them to the cavity surface, is currently being marketed. The main function is a final cleaning bed downstream of conventional demineralizing columns. Deionized water is used in pharmaceutical formulations, for washing containers and plant, and for the preparation of disinfectant solutions. 3.4 Distilled water As it leaves the still, distilled water is free from
Ecology of microorganisms as it affects the pharmaceutical industry
microorganisms, and contamination occurs as a result of a fault in the cooling system, the storage vessel or the distribution system. The flora of contaminated distilled water is usually Gram-negative bacteria and as it is introduced after a sterilization process, it is often a pure culture. A level of organisms up to 106 per ml has been recorded. Distilled water is often used in the formulation of oral and topical pharmaceutical preparations and a low bacterial count is desirable. It is also used after distillation with a specially designed still, often made of glass, for the manufacture of parenteral preparations and a post-distillation heat sterilization stage is commonly included in the process. Water for such preparations is often stored at 80°C to prevent bacterial growth and the production of pyrogenic substances which accompany such growth. 3.5 Water produced by reverse osmosis Water produced by reverse osmosis (RO) is forced by an osmotic pressure through a semi-permeable membrane which acts as a molecular filter. The diffusion of solubles dissolved in the water is impeded, and those with a molecular weight in excess of 250 do not diffuse at all. The process, which is the reverse of the natural process of osmosis, thus removes microorganisms and their pyrogens. Post-RO contamination may occur if the plant after the membrane, the storage vessel or the distribution system is not kept free from microorganisms. 3.6 Distribution system If microorganisms colonize a storage vessel, it then acts as a microbial reservoir and contaminates all water passing through it. It is therefore important that the contents of all storage vessels are tested regularly. Reservoirs of microorganisms may also build up in booster pumps, water meters and unused sections of pipeline. Where a high positive pressure is absent or cannot be continuously maintained, outlets such as cocks and taps may permit bacteria to enter the system. An optimum system for reducing the growth of microbial flora is one that ensures a constant recirculation of water at a positive pressure through a
ring-main without ‘dead-legs’ (areas which due to their location are not regularly used) and only very short branches to the take-off points. In addition there should be a system to re-sterilize the water, usually by membrane filtration or UV light treatment, just before return to the main storage tank. Some plumbing materials used for storage vessels, pipework and jointing may support microbial growth. Some plastics, in particular plasticized polyvinylchlorides and resins used in the manufacture of glass-reinforced plastics, have caused serious microbiological problems when used for water storage and distribution systems. Both natural and synthetic rubbers used for washers, O-rings and diaphragms are susceptible to contamination if not sanitized regularly. For jointing, packing and lubricating materials, PTFE and silicone-based compounds are superior to those based on natural products such as vegetable oils or fibres and animal fats, and petroleum-based compounds. 3.7 Disinfection of water Three methods are used for treating water, namely chemicals, filtration or light. 3.7.1 Chemical treatment Chemical treatment is applicable usually to raw, mains and softened water, but is also used to treat the storage and distribution systems of distilled and deionized water and of water produced by reverse osmosis (section 3.5). Sodium hypochlorite and chlorine gas are the most common agents for treating the water supply itself, and the concentration employed depends both upon the dwell time and the chlorine demand of the water. For most purposes a free residual chlorine level of 0.5–5 ppm is adequate. For storage vessels, pipelines, pumps and outlets a higher level of 50–100 ppm may be necessary, but it is usually necessary to use a descaling agent before disinfection in areas where the water is hard. Distilled, deionized and RO systems and pipelines may be treated with sodium hypochlorite or 1% formaldehyde solution. With deionized systems it is usual to exhaust the resin beds with brine before sterilization with formaldehyde to prevent its inactivation to 255
Chapter 15
paraformaldehyde. If only local contamination occurs, live steam is often effective in eradicating it. During chemical sterilization it is important that no ‘dead-legs’ remain untreated and that all instruments such as water meters are treated.
sodium thiosulphate. Although an incubation temperature of 37°C may be necessary to recover some pathogens or faecal contaminants from water, many indigenous species fail to grow at this temperature, and it is usual to incubate at 20–26°C for their detection.
3.7.2 Filtration Membrane filtration is useful where the usage is moderate and a continuous circulation of water can be maintained. Thus, with the exception of that drawn off for use, the water is continually being returned to the storage tank and refiltered. As many waterborne bacteria are small, it is usual to install a 0.22-µm pore-size membrane as the terminal filter and to use coarser prefilters to prolong its life. Membrane filters require regular sterilization to prevent microbial colonization and ‘grow through’. They may be treated chemically with the remainder of the storage/distribution system or removed and treated by moist heat. The latter method is usually the most successful for heavily contaminated filters. 3.7.3 Light UV light at a wavelength of 254 nm is useful for the disinfection of water of good optical clarity. Such treatment has an advantage over chemical disinfection as there is no odour or flavour problem and, unlike membrane filters, it is not subject to microbial colonization. One of the newer technologies suitable for disinfecting water is UV-rich high intensity light pulses in which 30% of the energy is at wavelengths of
